ijms-logo

Journal Browser

Journal Browser

Protein Folding

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (21 February 2023) | Viewed by 441590

Special Issue Editor


E-Mail Website
Guest Editor
Institut de Biotecnologia i de Biomedicina (IBB) and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
Interests: protein folding; protein aggregation; protein design; high-throughput screening; bioinformatics; amyloids; Parkinson; nanomaterials; phase separation; prions; drug discovery
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Protein folding is among the most complex and challenging processes in Biochemistry. It is accepted that, after the synthesis at the ribosome, most polypeptides must fold into their specific three-dimensional structures before they can exert any biological function. Only properly folded conformers can interact specifically with their molecular targets. Therefore, protein folding is central to many biological processes. It has long been known that the functional structure of a protein is coded by its primary one-dimensional amino acid sequence. Despite the molecular mechanisms behind the folding code are still not completely understood, it is also true that in recent years we have witnessed significant advances towards this goal, resulting from the development of new experimental approaches and sophisticated prediction methods, but specially arising from new ways of thinking about protein folding and dynamics. Folding within biomembranes, multi-domain protein folding, folding in the cell, molecular chaperone-assisted holding and the dynamics of intrinsically disordered proteins are among the newest and more active areas of research. Although protein folding and dynamics are behind virtually all cell reactions, ranging from transcription to motion, it is the link to human disease that has put this subject in the public eye, because protein misfolding and subsequent aggregation have been shown to be the cause underlying dozens of human disorders. It turns out that understanding and predicting protein folding would allow elucidating how misfolded proteins cause aggregation and cytotoxicity. Thus, in a scientific environment that promotes technology transfer and tends to leave basic since aside, it is encouraging to learn that first principles hold the clue for therapeutic intervention in devastating disorders such us Parkinson’s and Alzheimer’s diseases. 
The aim of this Special Issue is to illustrate, through selected works, frontier research in protein folding.

Prof. Dr. Salvador Ventura
Guest Editor

Submission

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. Papers will be published continuously (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are refereed through a peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed Open Access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1600 CHF.

Keywords

  • amyloid
  • calorimetry
  • chaperones
  • crowding and folding
  • downhill folding
  • energy lanscape
  • folding in biomembranes
  • folding in the cell
  • folding intermediates
  • folding kinetics
  • intrinsically disordered proteins
  • molecular dynamics simulation of folding
  • oxidative folding
  • protein aggregation
  • protein folding and design
  • protein folding and docking
  • protein folding and evolution
  • protein misfolding
  • single molecule folding
  • transition state analysis

Related Special Issues

Published Papers (65 papers)

Order results
Result details
Select all
Export citation of selected articles as:

Research

Jump to: Review, Other

21 pages, 3378 KiB  
Article
Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
by Veronika Džupponová, Nataša Tomášková, Andrea Antošová, Erik Sedlák and Gabriel Žoldák
Int. J. Mol. Sci. 2023, 24(7), 6787; https://doi.org/10.3390/ijms24076787 - 5 Apr 2023
Cited by 1 | Viewed by 1481
Abstract
Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at [...] Read more.
Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

15 pages, 1916 KiB  
Article
Intracellular Conformation of Amyotrophic Lateral Sclerosis-Causative TDP-43
by Akira Kitamura, Sachiko Yuno, Rintaro Kawamura and Masataka Kinjo
Int. J. Mol. Sci. 2023, 24(6), 5513; https://doi.org/10.3390/ijms24065513 - 14 Mar 2023
Viewed by 1551
Abstract
Transactive response element DNA/RNA-binding protein 43 kDa (TDP-43) is the causative protein of amyotrophic lateral sclerosis (ALS); several ALS-associated mutants of TDP-43 have been identified. TDP-43 has several domains: an N-terminal domain, two RNA/DNA-recognition motifs, and a C-terminal intrinsically disordered region (IDR). Its [...] Read more.
Transactive response element DNA/RNA-binding protein 43 kDa (TDP-43) is the causative protein of amyotrophic lateral sclerosis (ALS); several ALS-associated mutants of TDP-43 have been identified. TDP-43 has several domains: an N-terminal domain, two RNA/DNA-recognition motifs, and a C-terminal intrinsically disordered region (IDR). Its structures have been partially determined, but the whole structure remains elusive. In this study, we investigate the possible end-to-end distance between the N- and C-termini of TDP-43, its alterations due to ALS-associated mutations in the IDR, and its apparent molecular shape in live cells using Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). Furthermore, the interaction between ALS-associated TDP-43 and heteronuclear ribonucleoprotein A1 (hnRNP A1) is slightly stronger than that of wild-type TDP-43. Our findings provide insights into the structure of wild-type and ALS-associated mutants of TDP-43 in a cell. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

14 pages, 4141 KiB  
Article
Characterizing the Specific Recognition of Xanthurenic Acid by GEP1 and GEP1-GCα Interactions in cGMP Signaling Pathway in Gametogenesis of Malaria Parasites
by Cheng Zhu, Xiaoge Liang, Xu Chen, Miaomiao Liang, Jianting Zheng, Bingbing Wan and Shukun Luo
Int. J. Mol. Sci. 2023, 24(3), 2561; https://doi.org/10.3390/ijms24032561 - 29 Jan 2023
Cited by 1 | Viewed by 1628
Abstract
Gametogenesis is an essential step for malaria parasite transmission and is activated in mosquito by signals including temperature drop, pH change, and mosquito-derived xanthurenic acid (XA). Recently, a membrane protein gametogenesis essential protein 1 (GEP1) was found to be responsible for sensing these [...] Read more.
Gametogenesis is an essential step for malaria parasite transmission and is activated in mosquito by signals including temperature drop, pH change, and mosquito-derived xanthurenic acid (XA). Recently, a membrane protein gametogenesis essential protein 1 (GEP1) was found to be responsible for sensing these signals and interacting with a giant guanylate cyclase α (GCα) to activate the cGMP-PKG-Ca2+ signaling pathway for malaria parasite gametogenesis. However, the molecular mechanisms for this process remain unclear. In this study, we used AlphaFold2 to predict the structure of GEP1 and found that it consists of a conserved N-terminal helical domain and a transmembrane domain that adopts a structure similar to that of cationic amino acid transporters. Molecular docking results showed that XA binds to GEP1 via a pocket similar to the ligand binding sites of known amino acid transporters. In addition, truncations of this N-terminal sequence significantly enhanced the expression, solubility, and stability of GEP1. In addition, we found that GEP1 interacts with GCα via its C-terminal region, which is interrupted by mutations of a few conserved residues. These findings provide further insights into the molecular mechanism for the XA recognition by GEP1 and the activation of the gametogenesis of malaria parasites through GEP1-GCα interaction. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

13 pages, 4632 KiB  
Article
An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model
by Subbaian Brindha, Takahiro Yoshizue, Rawiwan Wongnak, Hitoshi Takemae, Mami Oba, Tetsuya Mizutani and Yutaka Kuroda
Int. J. Mol. Sci. 2022, 23(24), 15744; https://doi.org/10.3390/ijms232415744 - 12 Dec 2022
Cited by 4 | Viewed by 1700
Abstract
A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against [...] Read more.
A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD’s eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell’s receptor, with a binding constant of 7.9 × 10−9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

13 pages, 2489 KiB  
Article
REMD Simulations of Full-Length Alpha-Synuclein Together with Ligands Reveal Binding Region and Effect on Amyloid Conversion
by Pavel I. Semenyuk
Int. J. Mol. Sci. 2022, 23(19), 11545; https://doi.org/10.3390/ijms231911545 - 30 Sep 2022
Cited by 3 | Viewed by 1892
Abstract
Alpha-synuclein is a key protein involved in the development and progression of Parkinson’s disease and other synucleinopathies. The intrinsically disordered nature of alpha-synuclein hinders the computational screening of new drug candidates for the treatment of these neurodegenerative diseases. In the present work, replica [...] Read more.
Alpha-synuclein is a key protein involved in the development and progression of Parkinson’s disease and other synucleinopathies. The intrinsically disordered nature of alpha-synuclein hinders the computational screening of new drug candidates for the treatment of these neurodegenerative diseases. In the present work, replica exchange molecular dynamics simulations of the full-length alpha-synuclein together with low-molecular ligands were utilized to predict the binding site and effect on the amyloid-like conversion of the protein. This approach enabled an accurate prediction of the binding sites for three tested compounds (fasudil, phthalocyanine tetrasulfonate, and spermine), giving good agreement with data from experiments by other groups. Lots of information about the binding and protein conformational ensemble enabled the suggestion of a putative effect of the ligands on the amyloid-like conversion of alpha-synuclein and the mechanism of anti- and pro-amyloid activity of the tested compounds. Therefore, this approach looks promising for testing new drug candidates for binding with alpha-synuclein or other intrinsically disordered proteins and at the same time the estimation of the effect on protein behavior, including amyloid-like aggregation. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

17 pages, 2956 KiB  
Article
The Anfinsen Dogma: Intriguing Details Sixty-Five Years Later
by Giorgia Gambardella, Sara Notari, Dario Cavaterra, Federica Iavarone, Massimo Castagnola, Alessio Bocedi and Giorgio Ricci
Int. J. Mol. Sci. 2022, 23(14), 7759; https://doi.org/10.3390/ijms23147759 - 14 Jul 2022
Viewed by 1957
Abstract
The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role of the correct disulfide in the attainment of the native structure. [...] Read more.
The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role of the correct disulfide in the attainment of the native structure. CD analysis, mass spectrometry, fluorescence spectroscopy and enzyme activity indicate that native disulfides drive the formation of the secondary and tertiary structures that cannot be entirely formed in their absence. This opposes a common opinion that these structures are first formed and then stabilized by the native disulfides. Our results also indicate that a spontaneous re-oxidation of a reduced RNase cannot produce a complete recovery of activity, as described by many textbooks; this can be obtained only in the presence of a reshuffling solution such as GSH/GSSG. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

14 pages, 2133 KiB  
Article
Protein Folding Interdiction Strategy for Therapeutic Drug Development in Viral Diseases: Ebola VP40 and Influenza A M1
by Fernando Bergasa-Caceres and Herschel A. Rabitz
Int. J. Mol. Sci. 2022, 23(7), 3906; https://doi.org/10.3390/ijms23073906 - 31 Mar 2022
Cited by 2 | Viewed by 2545
Abstract
In a recent paper, we proposed the folding interdiction target region (FITR) strategy for therapeutic drug design in SARS-CoV-2. This paper expands the application of the FITR strategy by proposing therapeutic drug design approaches against Ebola virus disease and influenza A. We predict [...] Read more.
In a recent paper, we proposed the folding interdiction target region (FITR) strategy for therapeutic drug design in SARS-CoV-2. This paper expands the application of the FITR strategy by proposing therapeutic drug design approaches against Ebola virus disease and influenza A. We predict target regions for folding interdicting drugs on correspondingly relevant structural proteins of both pathogenic viruses: VP40 of Ebola, and matrix protein M1 of influenza A. Identification of the protein targets employs the sequential collapse model (SCM) for protein folding. It is explained that the model predicts natural peptide candidates in each case from which to start the search for therapeutic drugs. The paper also discusses how these predictions could be tested, as well as some challenges likely to be found when designing effective therapeutic drugs from the proposed peptide candidates. The FITR strategy opens a potential new avenue for the design of therapeutic drugs that promises to be effective against infectious diseases. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

12 pages, 2003 KiB  
Article
N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function
by Sonia Águila, Rosina Noto, Ginés Luengo-Gil, Salvador Espín, Nataliya Bohdan, María Eugenia de la Morena-Barrio, Julia Peñas, Maria Carmen Rodenas, Vicente Vicente, Javier Corral, Mauro Manno and Irene Martínez-Martínez
Int. J. Mol. Sci. 2021, 22(2), 516; https://doi.org/10.3390/ijms22020516 - 6 Jan 2021
Cited by 8 | Viewed by 3106
Abstract
N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its [...] Read more.
N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

15 pages, 2805 KiB  
Article
Ultra-Rapid Glutathionylation of Ribonuclease: Is This the Real Incipit of Its Oxidative Folding?
by Alessio Bocedi, Giada Cattani, Giorgia Gambardella, Silvia Ticconi, Flora Cozzolino, Ornella Di Fusco, Piero Pucci and Giorgio Ricci
Int. J. Mol. Sci. 2019, 20(21), 5440; https://doi.org/10.3390/ijms20215440 - 31 Oct 2019
Cited by 6 | Viewed by 2766
Abstract
Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast [...] Read more.
Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40–50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5’-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

19 pages, 3492 KiB  
Article
Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain
by Hye Min Lee, Soon Bin Kwon, Ahyun Son, Doo Hyun Kim, Kyun-Hwan Kim, Jonghyo Lim, Young-Guen Kwon, Jin Sun Kang, Byung Kyu Lee, Young Ho Byun and Baik L. Seong
Int. J. Mol. Sci. 2019, 20(11), 2847; https://doi.org/10.3390/ijms20112847 - 11 Jun 2019
Cited by 4 | Viewed by 4390
Abstract
Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are [...] Read more.
Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

12 pages, 2682 KiB  
Article
Quality Screening of Incorrectly Folded Soluble Aggregates from Functional Recombinant Proteins
by Soon Bin Kwon, Ji Eun Yu, Jihoon Kim, Hana Oh, Chan Park, Jinhee Lee and Baik L. Seong
Int. J. Mol. Sci. 2019, 20(4), 907; https://doi.org/10.3390/ijms20040907 - 19 Feb 2019
Cited by 6 | Viewed by 4724
Abstract
Solubility is the prime criterion for determining the quality of recombinant proteins, yet it often fails to represent functional activity due to the involvement of non-functional, misfolded, soluble aggregates, which compromise the quality of recombinant proteins. However, guidelines for the quality assessment of [...] Read more.
Solubility is the prime criterion for determining the quality of recombinant proteins, yet it often fails to represent functional activity due to the involvement of non-functional, misfolded, soluble aggregates, which compromise the quality of recombinant proteins. However, guidelines for the quality assessment of soluble proteins have neither been proposed nor rigorously validated experimentally. Using the aggregation-prone enhanced green-fluorescent protein (EGFP) folding reporter system, we evaluated the folding status of recombinant proteins by employing the commonly used sonication and mild lysis of recombinant host cells. We showed that the differential screening of solubility and folding competence is crucial for improving the quality of recombinant proteins without sacrificing their yield. These results highlight the importance of screening out incorrectly folded soluble aggregates at the initial purification step to ensure the functional quality of recombinant proteins. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

28 pages, 9161 KiB  
Article
A Hybrid Hamiltonian for the Accelerated Sampling along Experimental Restraints
by Emanuel K. Peter and Jiří Černý
Int. J. Mol. Sci. 2019, 20(2), 370; https://doi.org/10.3390/ijms20020370 - 16 Jan 2019
Cited by 4 | Viewed by 3877
Abstract
In this article, we present an enhanced sampling method based on a hybrid Hamiltonian which combines experimental distance restraints with a bias dependent from multiple path-dependent variables. This simulation method determines the bias-coordinates on the fly and does not require a priori knowledge [...] Read more.
In this article, we present an enhanced sampling method based on a hybrid Hamiltonian which combines experimental distance restraints with a bias dependent from multiple path-dependent variables. This simulation method determines the bias-coordinates on the fly and does not require a priori knowledge about reaction coordinates. The hybrid Hamiltonian accelerates the sampling of proteins, and, combined with experimental distance information, the technique considers the restraints adaptively and in dependency of the system’s intrinsic dynamics. We validate the methodology on the dipole relaxation of two water models and the conformational landscape of dialanine. Using experimental NMR-restraint data, we explore the folding landscape of the TrpCage mini-protein and in a second example apply distance restraints from chemical crosslinking/mass spectrometry experiments for the sampling of the conformation space of the Killer Cell Lectin-like Receptor Subfamily B Member 1A (NKR-P1A). The new methodology has the potential to adaptively introduce experimental restraints without affecting the conformational space of the system along an ergodic trajectory. Since only a limited number of input- and no-order parameters are required for the setup of the simulation, the method is broadly applicable and has the potential to be combined with coarse-graining methods. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

32 pages, 12021 KiB  
Article
Enriched Conformational Sampling of DNA and Proteins with a Hybrid Hamiltonian Derived from the Protein Data Bank
by Emanuel K. Peter and Jiří Černý
Int. J. Mol. Sci. 2018, 19(11), 3405; https://doi.org/10.3390/ijms19113405 - 30 Oct 2018
Cited by 3 | Viewed by 3380
Abstract
In this article, we present a method for the enhanced molecular dynamics simulation of protein and DNA systems called potential of mean force (PMF)-enriched sampling. The method uses partitions derived from the potentials of mean force, which we determined from DNA and protein [...] Read more.
In this article, we present a method for the enhanced molecular dynamics simulation of protein and DNA systems called potential of mean force (PMF)-enriched sampling. The method uses partitions derived from the potentials of mean force, which we determined from DNA and protein structures in the Protein Data Bank (PDB). We define a partition function from a set of PDB-derived PMFs, which efficiently compensates for the error introduced by the assumption of a homogeneous partition function from the PDB datasets. The bias based on the PDB-derived partitions is added in the form of a hybrid Hamiltonian using a renormalization method, which adds the PMF-enriched gradient to the system depending on a linear weighting factor and the underlying force field. We validated the method using simulations of dialanine, the folding of TrpCage, and the conformational sampling of the Dickerson–Drew DNA dodecamer. Our results show the potential for the PMF-enriched simulation technique to enrich the conformational space of biomolecules along their order parameters, while we also observe a considerable speed increase in the sampling by factors ranging from 13.1 to 82. The novel method can effectively be combined with enhanced sampling or coarse-graining methods to enrich conformational sampling with a partition derived from the PDB. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

15 pages, 3851 KiB  
Article
Nucleic Acid-Dependent Structural Transition of the Intrinsically Disordered N-Terminal Appended Domain of Human Lysyl-tRNA Synthetase
by Soon Bin Kwon, Ji Eun Yu, Chan Park, Jiseop Lee and Baik L. Seong
Int. J. Mol. Sci. 2018, 19(10), 3016; https://doi.org/10.3390/ijms19103016 - 3 Oct 2018
Cited by 11 | Viewed by 3541
Abstract
Eukaryotic lysyl-tRNA synthetases (LysRS) have an N-terminal appended tRNA-interaction domain (RID) that is absent in their prokaryotic counterparts. This domain is intrinsically disordered and lacks stable structures. The disorder-to-order transition is induced by tRNA binding and has implications on folding and subsequent assembly [...] Read more.
Eukaryotic lysyl-tRNA synthetases (LysRS) have an N-terminal appended tRNA-interaction domain (RID) that is absent in their prokaryotic counterparts. This domain is intrinsically disordered and lacks stable structures. The disorder-to-order transition is induced by tRNA binding and has implications on folding and subsequent assembly into multi-tRNA synthetase complexes. Here, we expressed and purified RID from human LysRS (hRID) in Escherichia coli and performed a detailed mutagenesis of the appended domain. hRID was co-purified with nucleic acids during Ni-affinity purification, and cumulative mutations on critical amino acid residues abolished RNA binding. Furthermore, we identified a structural ensemble between disordered and helical structures in non-RNA-binding mutants and an equilibrium shift for wild-type into the helical conformation upon RNA binding. Since mutations that disrupted RNA binding led to an increase in non-functional soluble aggregates, a stabilized RNA-mediated structural transition of the N-terminal appended domain may have implications on the functional organization of human LysRS and multi-tRNA synthetase complexes in vivo. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

11 pages, 3312 KiB  
Article
Visualization of Tau–Tubulin Interaction in a Living Cell Using Bifluorescence Complementation Technique
by Seulgi Shin, Sungsu Lim, Hyeanjeong Jeong, Li Ting Kwan and Yun Kyung Kim
Int. J. Mol. Sci. 2018, 19(10), 2978; https://doi.org/10.3390/ijms19102978 - 29 Sep 2018
Cited by 3 | Viewed by 4889
Abstract
Tau is a neuron-specific microtubule-binding protein that stabilizes microtubules. It is generally thought that highly phosphorylated tau dissociates from microtubules and becomes insoluble aggregates, leading to neuronal degeneration. Due to the implication of tau aggregation in neurodegenerative disorders, including Alzheimer’s disease, great efforts [...] Read more.
Tau is a neuron-specific microtubule-binding protein that stabilizes microtubules. It is generally thought that highly phosphorylated tau dissociates from microtubules and becomes insoluble aggregates, leading to neuronal degeneration. Due to the implication of tau aggregation in neurodegenerative disorders, including Alzheimer’s disease, great efforts have been made to identify the tau aggregation process. However, tau interaction with tubulin during the aggregation process remains largely unknown. To scrutinize the tau-tubulin interaction, we generated a cell model that enables visualization of the tau-tubulin interaction in a living cell using the Bifluorescence Complementation (BiFC) Technique. Upon diverse chemical stimulation that induced tau pathology, tau-tubulin BiFC cells showed significantly increased levels of BiFC fluorescence, indicating that tau aggregates together with tubulin. Our results suggest that tubulin should be considered as a key component in the tau aggregation process. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

13 pages, 2654 KiB  
Article
The Disordered C-Terminus of Yeast Hsf1 Contains a Cryptic Low-Complexity Amyloidogenic Region
by Jordi Pujols, Jaime Santos, Irantzu Pallarès and Salvador Ventura
Int. J. Mol. Sci. 2018, 19(5), 1384; https://doi.org/10.3390/ijms19051384 - 6 May 2018
Cited by 8 | Viewed by 4471
Abstract
Response mechanisms to external stress rely on networks of proteins able to activate specific signaling pathways to ensure the maintenance of cell proteostasis. Many of the proteins mediating this kind of response contain intrinsically disordered regions, which lack a defined structure, but still [...] Read more.
Response mechanisms to external stress rely on networks of proteins able to activate specific signaling pathways to ensure the maintenance of cell proteostasis. Many of the proteins mediating this kind of response contain intrinsically disordered regions, which lack a defined structure, but still are able to interact with a wide range of clients that modulate the protein function. Some of these interactions are mediated by specific short sequences embedded in the longer disordered regions. Because the physicochemical properties that promote functional and abnormal interactions are similar, it has been shown that, in globular proteins, aggregation-prone and binding regions tend to overlap. It could be that the same principle applies for disordered protein regions. In this context, we show here that a predicted low-complexity interacting region in the disordered C-terminus of the stress response master regulator heat shock factor 1 (Hsf1) protein corresponds to a cryptic amyloid region able to self-assemble into fibrillary structures resembling those found in neurodegenerative disorders. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

16 pages, 11196 KiB  
Article
Differential Glycosylation and Modulation of Camel and Human HSP Isoforms in Response to Thermal and Hypoxic Stresses
by Abdullah Hoter, Mahdi Amiri, Abdelbary Prince, Hassan Amer, Mohamad Warda and Hassan Y. Naim
Int. J. Mol. Sci. 2018, 19(2), 402; https://doi.org/10.3390/ijms19020402 - 30 Jan 2018
Cited by 13 | Viewed by 6562
Abstract
Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal [...] Read more.
Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

17 pages, 15288 KiB  
Article
Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by β-Synuclein
by Andre Leitao, Akshay Bhumkar, Dominic J. B. Hunter, Yann Gambin and Emma Sierecki
Int. J. Mol. Sci. 2018, 19(2), 334; https://doi.org/10.3390/ijms19020334 - 24 Jan 2018
Cited by 15 | Viewed by 5932
Abstract
α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as [...] Read more.
α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants—A30P and G51D—as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

11 pages, 1413 KiB  
Article
Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p
by Erik Sedlák, Gabriel Žoldák and Pernilla Wittung-Stafshede
Int. J. Mol. Sci. 2018, 19(1), 269; https://doi.org/10.3390/ijms19010269 - 16 Jan 2018
Cited by 3 | Viewed by 4785
Abstract
Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role [...] Read more.
Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

22 pages, 3471 KiB  
Article
Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients
by Marion Wattin, Loïc Gaweda, Pascale Muller, Mathieu Baritaud, Charlotte Scholtes, Chloé Lozano, Kathrin Gieseler and Carole Kretz-Remy
Int. J. Mol. Sci. 2018, 19(1), 178; https://doi.org/10.3390/ijms19010178 - 7 Jan 2018
Cited by 9 | Viewed by 5080
Abstract
The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular [...] Read more.
The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD) is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

3628 KiB  
Article
Insights into Insulin Fibril Assembly at Physiological and Acidic pH and Related Amyloid Intrinsic Fluorescence
by Clara Iannuzzi, Margherita Borriello, Marianna Portaccio, Gaetano Irace and Ivana Sirangelo
Int. J. Mol. Sci. 2017, 18(12), 2551; https://doi.org/10.3390/ijms18122551 - 28 Nov 2017
Cited by 61 | Viewed by 6344
Abstract
Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural [...] Read more.
Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, circular dichroism, Fourier-transform infrared spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphologically different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addition, only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally associated to amyloid fibrils. So far, the molecular origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide experimental evidence that allow identifying the molecular origin of such intrinsic property. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

4156 KiB  
Article
Effects of Metal Ions, Temperature, and a Denaturant on the Oxidative Folding Pathways of Bovine α-Lactalbumin
by Reina Shinozaki and Michio Iwaoka
Int. J. Mol. Sci. 2017, 18(9), 1996; https://doi.org/10.3390/ijms18091996 - 16 Sep 2017
Cited by 12 | Viewed by 4302
Abstract
Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61–77, 73–91) and des[6–120], which have two and three native SS bonds, respectively, accumulate predominantly in [...] Read more.
Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61–77, 73–91) and des[6–120], which have two and three native SS bonds, respectively, accumulate predominantly in the presence of Ca2+. In this study, we reinvestigated the pathways using a water-soluble cyclic selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHSox), as a strong and quantitative oxidant to oxidize the fully reduced form (R). In the presence of ethylenediaminetetraacetic acid (EDTA) (under a metal-free condition), SS formation randomly proceeded, and N did not regenerate. On the other hand, two specific SS intermediates transiently generated in the presence of Ca2+. These intermediates could be assigned to (61–77, 73–91) and des[6–120] having two common SS bonds, i.e., Cys61-Cys77 and Cys73-Cys91, near the calcium binding pocket of the β-sheet domain. Much faster folding to N was observed in the presence of Mn2+, whereas Na+, K+, Mg2+, and Zn2+ did not affect the pathways. The two key intermediates were susceptible to temperature and a denaturant. The oxidative folding pathways revealed were significantly different from those of hen egg white lysozyme, which has the same SS-bonding pattern as αLA, suggesting that the folding pathways of SS-containing proteins can alter depending on the amino acid sequence and other factors, even when the SS-bond topologies are similar to each other. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

3892 KiB  
Article
Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation
by Ajda Taler-Verčič, Samra Hasanbašić, Selma Berbić, Veronika Stoka, Dušan Turk and Eva Žerovnik
Int. J. Mol. Sci. 2017, 18(3), 549; https://doi.org/10.3390/ijms18030549 - 7 Mar 2017
Cited by 18 | Viewed by 11638
Abstract
Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin [...] Read more.
Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to β2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

12551 KiB  
Article
High-Throughput Screening Methodology to Identify Alpha-Synuclein Aggregation Inhibitors
by Jordi Pujols, Samuel Peña-Díaz, María Conde-Giménez, Francisca Pinheiro, Susanna Navarro, Javier Sancho and Salvador Ventura
Int. J. Mol. Sci. 2017, 18(3), 478; https://doi.org/10.3390/ijms18030478 - 2 Mar 2017
Cited by 55 | Viewed by 9326
Abstract
An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson’s disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent [...] Read more.
An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson’s disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

4216 KiB  
Article
Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu
by Olesya V. Stepanenko, Olga V. Stepanenko, Irina M. Kuznetsova, Vladimir N. Uversky and Konstantin K. Turoverov
Int. J. Mol. Sci. 2016, 17(11), 1805; https://doi.org/10.3390/ijms17111805 - 28 Oct 2016
Cited by 9 | Viewed by 5555
Abstract
The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly [...] Read more.
The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1460 KiB  
Article
A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
by Catarina S. H. Jesus, Zaida L. Almeida, Daniela C. Vaz, Tiago Q. Faria and Rui M. M. Brito
Int. J. Mol. Sci. 2016, 17(9), 1428; https://doi.org/10.3390/ijms17091428 - 31 Aug 2016
Cited by 6 | Viewed by 5981
Abstract
Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer’s and Parkinson’s. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid [...] Read more.
Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer’s and Parkinson’s. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

2163 KiB  
Article
Benzbromarone, Quercetin, and Folic Acid Inhibit Amylin Aggregation
by Laura C. López, Olga Varea, Susanna Navarro, José A. Carrodeguas, Natalia Sanchez de Groot, Salvador Ventura and Javier Sancho
Int. J. Mol. Sci. 2016, 17(6), 964; https://doi.org/10.3390/ijms17060964 - 18 Jun 2016
Cited by 35 | Viewed by 7678
Abstract
Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic β-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, [...] Read more.
Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic β-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, self-assembled into amyloid aggregates, and ultimately contributes to the apoptotic death of β-cells by mechanisms that are not completely understood. We have screened a library of approved drugs in order to identify inhibitors of amylin aggregation that could be used as tools to investigate the role of amylin aggregation in type II diabetes or as therapeutics in order to reduce β-cell damage. Interestingly, three of the compounds analyzed—benzbromarone, quercetin, and folic acid—are able to slow down amylin fiber formation according to Thioflavin T binding, turbidimetry, and Transmission Electron Microscopy assays. In addition to the in vitro assays, we have tested the effect of these compounds in an amyloid toxicity cell culture model and we have found that one of them, quercetin, has the ability to partly protect cultured pancreatic insulinoma cells from the cytotoxic effect of amylin. Our data suggests that quercetin can contribute to reduce oxidative damage in pancreatic insulinoma β cells by modulating the aggregation propensity of amylin. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Figure 1

5973 KiB  
Article
The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations
by Moye Wang, Jie Hu and Zhuqing Zhang
Int. J. Mol. Sci. 2016, 17(5), 612; https://doi.org/10.3390/ijms17050612 - 26 Apr 2016
Cited by 2 | Viewed by 6497
Abstract
As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, [...] Read more.
As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

3830 KiB  
Article
Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors
by Sungsu Lim, Md. Mamunul Haque, Ghilsoo Nam, Nayeon Ryoo, Hyewhon Rhim and Yun Kyung Kim
Int. J. Mol. Sci. 2015, 16(9), 20212-20224; https://doi.org/10.3390/ijms160920212 - 26 Aug 2015
Cited by 36 | Viewed by 10302
Abstract
Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. [...] Read more.
Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

2539 KiB  
Article
Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly
by Steven Blake, Samiul Amin, Wei Qi, Madhabi Majumdar and E. Neil Lewis
Int. J. Mol. Sci. 2015, 16(8), 17719-17733; https://doi.org/10.3390/ijms160817719 - 3 Aug 2015
Cited by 11 | Viewed by 6884
Abstract
A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS), optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC). The diffusion [...] Read more.
A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS), optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC). The diffusion interaction parameter kD emerges as an accurate predictor of colloidal stability for this protein consistent with observed aggregation trends and rheology. Drastic aggregation and gelation were observed at pH 5.5. Under this condition, the protein’s secondary and tertiary structures changed simultaneously. At higher pH (7.0 and 8.5), oligomerizaton with no gel formation occurred. For these solutions, tertiary structure and secondary structure transitions were sequential. The low frequency Raman data, which is a good indicator of hydrogen bonding and structuring in water, has been shown to exhibit a strong correlation with the rheological evolution with temperature. This study has, for the first time, demonstrated that this low frequency Raman data, in conjunction with the DSC endotherm, can be been utilized to deconvolve protein unfolding and aggregation/gelation. These findings can have important implications for the development of protein-based biotherapeutics, where the formulation viscosity, aggregation, and stability strongly affects efficacy or in foods where protein structuring is critical for functional and sensory performance. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1481 KiB  
Article
A Multi-Objective Approach for Protein Structure Prediction Based on an Energy Model and Backbone Angle Preferences
by Jyh-Jong Tsay, Shih-Chieh Su and Chin-Sheng Yu
Int. J. Mol. Sci. 2015, 16(7), 15136-15149; https://doi.org/10.3390/ijms160715136 - 3 Jul 2015
Cited by 4 | Viewed by 5696
Abstract
Protein structure prediction (PSP) is concerned with the prediction of protein tertiary structure from primary structure and is a challenging calculation problem. After decades of research effort, numerous solutions have been proposed for optimisation methods based on energy models. However, further investigation and [...] Read more.
Protein structure prediction (PSP) is concerned with the prediction of protein tertiary structure from primary structure and is a challenging calculation problem. After decades of research effort, numerous solutions have been proposed for optimisation methods based on energy models. However, further investigation and improvement is still needed to increase the accuracy and similarity of structures. This study presents a novel backbone angle preference factor, which is one of the factors inducing protein folding. The proposed multiobjective optimisation approach simultaneously considers energy models and backbone angle preferences to solve the ab initio PSP. To prove the effectiveness of the multiobjective optimisation approach based on the energy models and backbone angle preferences, 75 amino acid sequences with lengths ranging from 22 to 88 amino acids were selected from the CB513 data set to be the benchmarks. The data sets were highly dissimilar, therefore indicating that they are meaningful. The experimental results showed that the root-mean-square deviation (RMSD) of the multiobjective optimization approach based on energy model and backbone angle preferences was superior to those of typical energy models, indicating that the proposed approach can facilitate the ab initio PSP. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1696 KiB  
Article
Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics
by Hyung-Nam Song, Jun-Hyuck Jang, Young-Wan Kim, Dong-Hyung Kim, Sung-Goo Park, Myung Kyu Lee, Se-Hwan Paek and Eui-Jeon Woo
Int. J. Mol. Sci. 2014, 15(12), 23658-23671; https://doi.org/10.3390/ijms151223658 - 18 Dec 2014
Cited by 12 | Viewed by 10401
Abstract
Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage [...] Read more.
Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VHVL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

677 KiB  
Article
Thermal Stability Threshold for Amyloid Formation in Light Chain Amyloidosis
by Tanya L. Poshusta, Nagaaki Katoh, Morie A. Gertz, Angela Dispenzieri and Marina Ramirez-Alvarado
Int. J. Mol. Sci. 2013, 14(11), 22604-22617; https://doi.org/10.3390/ijms141122604 - 15 Nov 2013
Cited by 24 | Viewed by 7188
Abstract
Light chain (AL) amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients [...] Read more.
Light chain (AL) amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients who achieve hematological complete response (CR) do not necessarily achieve organ response regardless of the treatment they received. In order to investigate the possible correlation between amyloid formation kinetics and organ response, we selected AL amyloidosis patients from the trial with kidney involvement and CR after treatment. Six patients were selected and their monoclonal immunoglobulin light chains were characterized. The proteins showed differences in their stability and their kinetics of amyloid formation. A correlation was detected at pH 7.4, showing that less stable proteins are more likely to form amyloid fibrils. AL-T03 is too unstable to form amyloid fibrils at pH 7.4. This protein was found in the only patient in the study that had organ response, suggesting that partially folded species are required for amyloid formation to occur in AL amyloidosis. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

906 KiB  
Article
Small Molecules Present in the Cerebrospinal Fluid Metabolome Influence Superoxide Dismutase 1 Aggregation
by Joana S. Cristóvão, Sónia S. Leal, Isabel Cardoso and Cláudio M. Gomes
Int. J. Mol. Sci. 2013, 14(9), 19128-19145; https://doi.org/10.3390/ijms140919128 - 17 Sep 2013
Cited by 5 | Viewed by 7363
Abstract
Superoxide dismutase 1 (SOD1) aggregation is one of the pathological markers of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The underlying molecular grounds of SOD1 pathologic aggregation remains obscure as mutations alone are not exclusively the cause for the formation of protein [...] Read more.
Superoxide dismutase 1 (SOD1) aggregation is one of the pathological markers of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The underlying molecular grounds of SOD1 pathologic aggregation remains obscure as mutations alone are not exclusively the cause for the formation of protein inclusions. Thus, other components in the cell environment likely play a key role in triggering SOD1 toxic aggregation in ALS. Recently, it was found that ALS patients present a specific altered metabolomic profile in the cerebrospinal fluid (CSF) where SOD1 is also present and potentially interacts with metabolites. Here we have investigated how some of these small molecules affect apoSOD1 structure and aggregation propensity. Our results show that as co-solvents, the tested small molecules do not affect apoSOD1 thermal stability but do influence its tertiary interactions and dynamics, as evidenced by combined biophysical analysis and proteolytic susceptibility. Moreover, these compounds influence apoSOD1 aggregation, decreasing nucleation time and promoting the formation of larger and less soluble aggregates, and in some cases polymeric assemblies apparently composed by spherical species resembling the soluble native protein. We conclude that some components of the ALS metabolome that shape the chemical environment in the CSF may influence apoSOD1 conformers and aggregation. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1436 KiB  
Article
The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B
by Ajda Taler-Verčič, Tiina Kirsipuu, Merlin Friedemann, Andra Noormägi, Mira Polajnar, Julia Smirnova, Magda Tušek Žnidarič, Matjaž Žganec, Miha Škarabot, Andrej Vilfan, Rosemary A. Staniforth, Peep Palumaa and Eva Žerovnik
Int. J. Mol. Sci. 2013, 14(9), 18362-18384; https://doi.org/10.3390/ijms140918362 - 5 Sep 2013
Cited by 12 | Viewed by 10037
Abstract
Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human [...] Read more.
Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

953 KiB  
Article
Liberation of GPI-Anchored Prion from Phospholipids Accelerates Amyloidogenic Conversion
by Shen-Jie Lin, Kun-Hua Yu, Jhih-Ru Wu, Chin-Fa Lee, Cheng-Ping Jheng, Hau-Ren Chen and Cheng-I Lee
Int. J. Mol. Sci. 2013, 14(9), 17943-17957; https://doi.org/10.3390/ijms140917943 - 3 Sep 2013
Cited by 5 | Viewed by 6522
Abstract
Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and [...] Read more.
Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1802 KiB  
Article
Trifluoroethanol Modulates Amyloid Formation by the All α-Helical URN1 FF Domain
by Patrizia Marinelli, Virginia Castillo and Salvador Ventura
Int. J. Mol. Sci. 2013, 14(9), 17830-17844; https://doi.org/10.3390/ijms140917830 - 30 Aug 2013
Cited by 10 | Viewed by 6828
Abstract
Amyloid fibril formation is implicated in different human diseases. The transition between native α-helices and nonnative intermolecular β-sheets has been suggested to be a trigger of fibrillation in different conformational diseases. The FF domain of the URN1 splicing factor (URN1-FF) is a small [...] Read more.
Amyloid fibril formation is implicated in different human diseases. The transition between native α-helices and nonnative intermolecular β-sheets has been suggested to be a trigger of fibrillation in different conformational diseases. The FF domain of the URN1 splicing factor (URN1-FF) is a small all-α protein that populates a molten globule (MG) at low pH. Despite the fact that this conformation maintains most of the domain native secondary structure, it progressively converts into β-sheet enriched and highly ordered amyloid fibrils. In this study, we investigated if 2,2,2-trifluoroethanol (TFE) induced conformational changes that affect URN1-FF amyloid formation. Despite TFE having been shown to induce or increase the aggregation of both globular and disordered proteins at moderate concentrations, we demonstrate here that in the case of URN1-FF it reinforces its intrinsic α-helical structure, which competes the formation of aggregated assemblies. In addition, we show that TFE induces conformational diversity in URN1-FF fibrils, in such a way that the fibrils formed in the presence and absence of the cosolvent represent different polymorphs. It is suggested that the effect of TFE on both the soluble and aggregated states of URN1-FF depends on its ability to facilitate hydrogen bonding. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

3168 KiB  
Article
Assessing the Effect of Loop Mutations in the Folding Space of β2-Microglobulin with Molecular Dynamics Simulations
by Sílvia G. Estácio, Eugene I. Shakhnovich and Patrícia F. N. Faísca
Int. J. Mol. Sci. 2013, 14(9), 17256-17278; https://doi.org/10.3390/ijms140917256 - 22 Aug 2013
Cited by 14 | Viewed by 8428
Abstract
We use molecular dynamics simulations of a full atomistic Gō model to explore the impact of selected DE-loop mutations (D59P and W60C) on the folding space of protein human β2-microglobulin (Hβ2m), the causing agent of dialysis-related amyloidosis, a conformational [...] Read more.
We use molecular dynamics simulations of a full atomistic Gō model to explore the impact of selected DE-loop mutations (D59P and W60C) on the folding space of protein human β2-microglobulin (Hβ2m), the causing agent of dialysis-related amyloidosis, a conformational disorder characterized by the deposition of insoluble amyloid fibrils in the osteoarticular system. Our simulations replicate the effect of mutations on the thermal stability that is observed in experiments in vitro. Furthermore, they predict the population of a partially folded state, with 60% of native internal free energy, which is akin to a molten globule. In the intermediate state, the solvent accessible surface area increases up to 40 times relative to the native state in 38% of the hydrophobic core residues, indicating that the identified species has aggregation potential. The intermediate state preserves the disulfide bond established between residue Cys25 and residue Cys80, which helps maintain the integrity of the core region, and is characterized by having two unstructured termini. The movements of the termini dominate the essential modes of the intermediate state, and exhibit the largest displacements in the D59P mutant, which is the most aggregation prone variant. PROPKA predictions of pKa suggest that the population of the intermediate state may be enhanced at acidic pH explaining the larger amyloidogenic potential observed in vitro at low pH for the WT protein and mutant forms. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

841 KiB  
Article
Transition Pathway and Its Free-Energy Profile: A Protocol for Protein Folding Simulations
by In-Ho Lee, Seung-Yeon Kim and Jooyoung Lee
Int. J. Mol. Sci. 2013, 14(8), 16058-16075; https://doi.org/10.3390/ijms140816058 - 2 Aug 2013
Cited by 3 | Viewed by 7352
Abstract
We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD), we investigate the dynamic folding pathway model of a protein between a [...] Read more.
We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD), we investigate the dynamic folding pathway model of a protein between a fixed extended conformation and a compact conformation. We choose the pathway model to be the reaction coordinate, and the folding and unfolding processes are characterized by the ADMD step index, in contrast to the common a priori reaction coordinate as used in conventional studies. Second, we calculate free-energy profile as the function of temperature, by employing the replica-exchange molecular dynamics (REMD) method. The current method provides efficient exploration of conformational space and proper characterization of protein folding/unfolding dynamics from/to an arbitrary extended conformation. We demonstrate that combination of the two simulation methods, ADMD and REMD, provides understanding on molecular conformational changes in proteins. The protocol is tested on a small protein, penta-peptide of met-enkephalin. For the neuropeptide met-enkephalin system, folded, extended, and intermediate sates are well-defined through the free-energy profile over the reaction coordinate. Results are consistent with those in the literature. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

1137 KiB  
Article
Structure Prediction of Partial-Length Protein Sequences
by Adrian Laurenzi, Ling-Hong Hung and Ram Samudrala
Int. J. Mol. Sci. 2013, 14(7), 14892-14907; https://doi.org/10.3390/ijms140714892 - 17 Jul 2013
Cited by 7 | Viewed by 6785
Abstract
Protein structure information is essential to understand protein function. Computational methods to accurately predict protein structure from the sequence have primarily been evaluated on protein sequences representing full-length native proteins. Here, we demonstrate that top-performing structure prediction methods can accurately predict the partial [...] Read more.
Protein structure information is essential to understand protein function. Computational methods to accurately predict protein structure from the sequence have primarily been evaluated on protein sequences representing full-length native proteins. Here, we demonstrate that top-performing structure prediction methods can accurately predict the partial structures of proteins encoded by sequences that contain approximately 50% or more of the full-length protein sequence. We hypothesize that structure prediction may be useful for predicting functions of proteins whose corresponding genes are mapped expressed sequence tags (ESTs) that encode partial-length amino acid sequences. Additionally, we identify a confidence score representing the quality of a predicted structure as a useful means of predicting the likelihood that an arbitrary polypeptide sequence represents a portion of a foldable protein sequence (“foldability”). This work has ramifications for the prediction of protein structure with limited or noisy sequence information, as well as genome annotation. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

1159 KiB  
Article
Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control
by Kenta Arai, Wataru Shibagaki, Reina Shinozaki and Michio Iwaoka
Int. J. Mol. Sci. 2013, 14(7), 13194-13212; https://doi.org/10.3390/ijms140713194 - 26 Jun 2013
Cited by 17 | Viewed by 6347
Abstract
It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76–94], des[64–80], and des [6–127], are populated at 20 °C and N [...] Read more.
It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76–94], des[64–80], and des [6–127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64–80] and des[6–127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5–45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When reduced HEL was reacted with 1–4 equivalents of DHSox, 1S, 2S, 3S, and 4S intermediate ensembles with 1–4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76–94] was populated in the largest amount, but the oxidation to N was slower than that of des[64–80] and des[6–127]. At 35 °C, on the other hand, des[64–80] and des[6–127] were no longer stable, and only des[76–94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

772 KiB  
Article
Tracking the Interplay between Bound Peptide and the Lid Domain of DnaK, Using Molecular Dynamics
by Itzhaq Azoulay, Nataly Kucherenko, Esther Nachliel, Menachem Gutman, Abdussalam Azem and Yossi Tsfadia
Int. J. Mol. Sci. 2013, 14(6), 12675-12695; https://doi.org/10.3390/ijms140612675 - 17 Jun 2013
Cited by 4 | Viewed by 7739
Abstract
Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD), that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD), which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of [...] Read more.
Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD), that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD), which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of the SBD of the bacterial Hsp70, DnaK, consists of two sub-domains: A β-sandwich part containing the hydrophobic cavity to which the hepta-peptide NRLLLTG (NR) is bound, and a segment made of 5 α-helices, called the “lid” that caps the top of the β-sandwich domain. In the present study we used the Escherichia coli Hsp70, DnaK, as a model for Hsp70 proteins, focusing on its SBD domain, examining the changes in the lid conformation. We deliberately decoupled the NBD from the SBD, limiting the study to the structure of the SBD section, with an emphasis on the interaction between the charges of the peptide with the residues located in the lid. Molecular dynamics simulations of the complex revealed significant mobility within the lid structure; as the structure was released from the forces operating during the crystallization process, the two terminal helices established a contact with the positive charge at the tip of the peptide. This contact is manifested only in the presence of electrostatic attraction. The observed internal motions within the lid provide a molecular role for the function of this sub-domain during the reaction cycle of Hsp 70 chaperones. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1790 KiB  
Article
A Hamiltonian Replica Exchange Molecular Dynamics (MD) Method for the Study of Folding, Based on the Analysis of the Stabilization Determinants of Proteins
by Massimiliano Meli and Giorgio Colombo
Int. J. Mol. Sci. 2013, 14(6), 12157-12169; https://doi.org/10.3390/ijms140612157 - 6 Jun 2013
Cited by 22 | Viewed by 9962
Abstract
Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, [...] Read more.
Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, the compact energetic map of the native state is generated by a preliminary short molecular dynamics (MD) simulation of the protein in explicit solvent. This map is simplified by means of an eigenvalue decomposition. The highest components of the eigenvector associated with the lowest eigenvalue indicate which sites, named “hot spots”, are likely to be responsible for the stability and correct folding of the protein. In the Hamiltonian replica exchange protocol, we use modified force-field parameters to treat the interparticle non-bonded potentials of the hot spots within the protein and between protein and solvent atoms, leaving unperturbed those relative to all other residues, as well as solvent-solvent interactions. We show that it is possible to reversibly simulate the folding/unfolding behavior of two test proteins, namely Villin HeadPiece HP35 (35 residues) and Protein A (62 residues), using a limited number of replicas. We next discuss possible implications for the study of folding mechanisms via all atom simulations. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

1902 KiB  
Article
Turn-Directed α-β Conformational Transition of α-syn12 Peptide at Different pH Revealed by Unbiased Molecular Dynamics Simulations
by Lei Liu and Zanxia Cao
Int. J. Mol. Sci. 2013, 14(6), 10896-10907; https://doi.org/10.3390/ijms140610896 - 24 May 2013
Cited by 5 | Viewed by 7421
Abstract
The transition from α-helical to β-hairpin conformations of α-syn12 peptide is characterized here using long timescale, unbiased molecular dynamics (MD) simulations in explicit solvent models at physiological and acidic pH values. Four independent normal MD trajectories, each 2500 ns, are performed at 300 [...] Read more.
The transition from α-helical to β-hairpin conformations of α-syn12 peptide is characterized here using long timescale, unbiased molecular dynamics (MD) simulations in explicit solvent models at physiological and acidic pH values. Four independent normal MD trajectories, each 2500 ns, are performed at 300 K using the GROMOS 43A1 force field and SPC water model. The most clustered structures at both pH values are β-hairpin but with different turns and hydrogen bonds. Turn9-6 and four hydrogen bonds (HB9-6, HB6-9, HB11-4 and HB4-11) are formed at physiological pH; turn8-5 and five hydrogen bonds (HB8-5, HB5-8, HB10-3, HB3-10 and HB12-1) are formed at acidic pH. A common folding mechanism is observed: the formation of the turn is always before the formation of the hydrogen bonds, which means the turn is always found to be the major determinant in initiating the transition process. Furthermore, two transition paths are observed at physiological pH. One of the transition paths tends to form the most-clustered turn and improper hydrogen bonds at the beginning, and then form the most-clustered hydrogen bonds. Another transition path tends to form the most-clustered turn, and turn5-2 firstly, followed by the formation of part hydrogen bonds, then turn5-2 is extended and more hydrogen bonds are formed. The transition path at acidic pH is as the same as the first path described at physiological pH. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

407 KiB  
Article
Combining Coarse-Grained Protein Models with Replica-Exchange All-Atom Molecular Dynamics
by Jacek Wabik, Sebastian Kmiecik, Dominik Gront, Maksim Kouza and Andrzej Koliński
Int. J. Mol. Sci. 2013, 14(5), 9893-9905; https://doi.org/10.3390/ijms14059893 - 10 May 2013
Cited by 18 | Viewed by 7943
Abstract
We describe a combination of all-atom simulations with CABS, a well-established coarse-grained protein modeling tool, into a single multiscale protocol. The simulation method has been tested on the C-terminal beta hairpin of protein G, a model system of protein folding. After reconstructing atomistic [...] Read more.
We describe a combination of all-atom simulations with CABS, a well-established coarse-grained protein modeling tool, into a single multiscale protocol. The simulation method has been tested on the C-terminal beta hairpin of protein G, a model system of protein folding. After reconstructing atomistic details, conformations derived from the CABS simulation were subjected to replica-exchange molecular dynamics simulations with OPLS-AA and AMBER99sb force fields in explicit solvent. Such a combination accelerates system convergence several times in comparison with all-atom simulations starting from the extended chain conformation, demonstrated by the analysis of melting curves, the number of native-like conformations as a function of time and secondary structure propagation. The results strongly suggest that the proposed multiscale method could be an efficient and accurate tool for high-resolution studies of protein folding dynamics in larger systems. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1184 KiB  
Article
Recurrent Structural Motifs in Non-Homologous Protein Structures
by Maria U. Johansson, Vincent Zoete and Nicolas Guex
Int. J. Mol. Sci. 2013, 14(4), 7795-7814; https://doi.org/10.3390/ijms14047795 - 10 Apr 2013
Cited by 3 | Viewed by 6640
Abstract
We have extracted an extensive collection of recurrent structural motifs (RSMs), which consist of sequentially non-contiguous structural motifs (4–6 residues), each of which appears with very similar conformation in three or more mutually unrelated protein structures. We find that the proteins in our [...] Read more.
We have extracted an extensive collection of recurrent structural motifs (RSMs), which consist of sequentially non-contiguous structural motifs (4–6 residues), each of which appears with very similar conformation in three or more mutually unrelated protein structures. We find that the proteins in our set are covered to a substantial extent by the recurrent non-contiguous structural motifs, especially the helix and strand regions. Computational alanine scanning calculations indicate that the average folding free energy changes upon alanine mutation for most types of non-alanine residues are higher for amino acids that are present in recurrent structural motifs than for amino acids that are not. The non-alanine amino acids that are most common in the recurrent structural motifs, i.e., phenylalanine, isoleucine, leucine, valine and tyrosine and the less abundant methionine and tryptophan, have the largest folding free energy changes. This indicates that the recurrent structural motifs, as we define them, describe recurrent structural patterns that are important for protein stability. In view of their properties, such structural motifs are potentially useful for inter-residue contact prediction and protein structure refinement. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

1822 KiB  
Article
Adsorption and Orientation of Human Islet Amyloid Polypeptide (hIAPP) Monomer at Anionic Lipid Bilayers: Implications for Membrane-Mediated Aggregation
by Yan Jia, Zhenyu Qian, Yun Zhang and Guanghong Wei
Int. J. Mol. Sci. 2013, 14(3), 6241-6258; https://doi.org/10.3390/ijms14036241 - 19 Mar 2013
Cited by 33 | Viewed by 8655
Abstract
Protein misfolding and aggregation cause serious degenerative diseases, such as Alzheimer’s and type II diabetes. Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits found in the pancreas of type II diabetic patients. Increasing evidence suggests that β-cell death is [...] Read more.
Protein misfolding and aggregation cause serious degenerative diseases, such as Alzheimer’s and type II diabetes. Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits found in the pancreas of type II diabetic patients. Increasing evidence suggests that β-cell death is related to the interaction of hIAPP with the cellular membrane, which accelerates peptide aggregation. In this study, as a first step towards understanding the membrane-mediated hIAPP aggregation, we investigate the atomic details of the initial step of hIAPP-membrane interaction, including the adsorption orientation and conformation of hIAPP monomer at an anionic POPG lipid bilayer by performing all-atom molecular dynamics simulations. We found that hIAPP monomer is quickly adsorbed to bilayer surface, and the adsorption is initiated from the N-terminal residues driven by strong electrostatic interactions of the positively-charged residues K1 and R11 with negatively-charged lipid headgroups. hIAPP binds parallel to the lipid bilayer surface as a stable helix through residues 7–22, consistent with previous experimental study. Remarkably, different simulations lead to the same binding orientation stabilized by electrostatic and H-bonding interactions, with residues R11, F15 and S19 oriented towards membrane and hydrophobic residues L12, A13, L16 and V17 exposed to solvent. Implications for membrane-mediated hIAPP aggregation are discussed. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

4307 KiB  
Article
Inhibition of Human Transthyretin Aggregation by Non-Steroidal Anti-Inflammatory Compounds: A Structural and Thermodynamic Analysis
by Ricardo O. Sant'Anna, Carolina A. Braga, Igor Polikarpov, Salvador Ventura, Luis Mauricio T. R. Lima and Debora Foguel
Int. J. Mol. Sci. 2013, 14(3), 5284-5311; https://doi.org/10.3390/ijms14035284 - 6 Mar 2013
Cited by 18 | Viewed by 8310
Abstract
Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are [...] Read more.
Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are composed of WT-TTR, while in FAP more than 100 already-described variants have been found in deposits. Until now, TTR-related diseases have been untreatable, although a new drug called Tafamidis has been approved only in Europe to specifically treat V30M patients. Thus, new strategies are still necessary to treat FAP caused by other variants of TTR. TTR has two channels in the dimer interface that bind to the hormone thyroxin and that have been used to accommodate anti-amyloidogenic compounds. These compounds stabilize the tetramers, rendering TTR less amyloidogenic. Here, we investigated the effects of three non-steroidal anti-inflammatory compounds—sulindac (SUL), indomethacin (IND) and lumiracoxib (LUM)—as tetramer stabilizers and aggregation inhibitors. WT-TTR and the very aggressive TTR variant L55P were used as models. These compounds were able to stabilize TTR against high hydrostatic pressure (HHP), increasing the ΔGf by several kcal. They were also effective in inhibiting WT-TTR and L55P acid- or HHP-induced aggregation; in particular, LUM and IND were very effective, inhibiting almost 100% of the aggregation of both proteins under certain conditions. The species formed when aggregation was performed in the presence of these compounds were much less toxic to cells in culture. The crystal structures of WT-TTR bound to the three compounds were solved at high resolution, allowing the identification of the relevant protein:drug interactions. We discuss here the ligand-binding features of LUM, IND and SUL to TTR, emphasizing the critical interactions that render the protein more stable and less amyloidogenic. Full article
(This article belongs to the Special Issue Protein Folding)
Show Figures

Graphical abstract

Review

Jump to: Research, Other

25 pages, 1332 KiB  
Review
The Role of Small Heat Shock Proteins in Protein Misfolding Associated Motoneuron Diseases
by