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18 pages, 3439 KiB

Open AccessArticle

Modified Protein-Water Interactions in CHARMM36m for Thermodynamics and Kinetics of Proteins in Dilute and Crowded Solutions

by Daiki Matsubara, Kento Kasahara, Hisham M. Dokainish, Hiraku Oshima and Yuji Sugita

Molecules 2022, 27(17), 5726; https://doi.org/10.3390/molecules27175726 - 05 Sep 2022

Cited by 6 | Viewed by 2176

Abstract

Proper balance between protein-protein and protein-water interactions is vital for atomistic molecular dynamics (MD) simulations of globular proteins as well as intrinsically disordered proteins (IDPs). The overestimation of protein-protein interactions tends to make IDPs more compact than those in experiments. Likewise, multiple proteins [...] Read more.

Proper balance between protein-protein and protein-water interactions is vital for atomistic molecular dynamics (MD) simulations of globular proteins as well as intrinsically disordered proteins (IDPs). The overestimation of protein-protein interactions tends to make IDPs more compact than those in experiments. Likewise, multiple proteins in crowded solutions are aggregated with each other too strongly. To optimize the balance, Lennard-Jones (LJ) interactions between protein and water are often increased about 10% (with a scaling parameter, λ = 1.1) from the existing force fields. Here, we explore the optimal scaling parameter of protein-water LJ interactions for CHARMM36m in conjunction with the modified TIP3P water model, by performing enhanced sampling MD simulations of several peptides in dilute solutions and conventional MD simulations of globular proteins in dilute and crowded solutions. In our simulations, 10% increase of protein-water LJ interaction for the CHARMM36m cannot maintain stability of a small helical peptide, (AAQAA)₃ in a dilute solution and only a small modification of protein-water LJ interaction up to the 3% increase (λ = 1.03) is allowed. The modified protein-water interactions are applicable to other peptides and globular proteins in dilute solutions without changing thermodynamic properties from the original CHARMM36m. However, it has a great impact on the diffusive properties of proteins in crowded solutions, avoiding the formation of too sticky protein-protein interactions. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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13 pages, 2753 KiB

Open AccessArticle

Interdiction in the Early Folding of the p53 DNA-Binding Domain Leads to Its Amyloid-Like Misfolding

by Fernando Bergasa-Caceres and Herschel A. Rabitz

Molecules 2022, 27(15), 4810; https://doi.org/10.3390/molecules27154810 - 27 Jul 2022

Viewed by 1553

Abstract

In this article, we investigate two issues: (a) the initial contact formation events along the folding pathway of the DNA-binding domain of the tumor suppressor protein p53 (core p53); and (b) the intermolecular events leading to its conversion into a prion-like form upon [...] Read more.

In this article, we investigate two issues: (a) the initial contact formation events along the folding pathway of the DNA-binding domain of the tumor suppressor protein p53 (core p53); and (b) the intermolecular events leading to its conversion into a prion-like form upon incubation with peptide P8(250-257). In the case of (a), the calculations employ the sequential collapse model (SCM) to identify the segments involved in the initial contact formation events that nucleate the folding pathway. The model predicts that there are several possible initial non-local contacts of comparative stability. The most stable of these possible initial contacts involve the protein segments ¹⁵⁹AMAIY¹⁶³ and ²⁵¹ILTII^255, and it is the only native-like contact. Thus, it is predicted to constitute “Nature’s shortcut” to the native structure of the core domain of p53. In the case of issue (b), these findings are then combined with experimental evidence showing that the incubation of the core domain of p53 with peptide P8(250-257), which is equivalent to the native protein segment ²⁵⁰PILTIITL²⁵⁷, leads to an amyloid conformational transition. It is explained how the SCM predicts that P8(250-257) effectively interdicts in the formation of the most stable possible initial contact and, thereby, disrupts the subsequent normal folding. Interdiction by polymeric P8(250-257) seeds is also studied. It is then hypothesized that enhanced folding through one or several of the less stable contacts could play a role in P8(250-257)-promoted core p53 amyloid misfolding. These findings are compared to previous results obtained for the prion protein. Experiments are proposed to test the hypothesis presented regarding core p53 amyloid misfolding. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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22 pages, 3383 KiB

Open AccessArticle

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity

by Matteo Moretti, Isabella Marzi, Cristina Cantarutti, Mirella Vivoli Vega, Walter Mandaliti, Maria Chiara Mimmi, Francesco Bemporad, Alessandra Corazza and Fabrizio Chiti

Molecules 2022, 27(13), 4309; https://doi.org/10.3390/molecules27134309 - 05 Jul 2022

Cited by 2 | Viewed by 2283

Abstract

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation [...] Read more.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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14 pages, 2421 KiB

Open AccessArticle

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain

by Yuki Yoshikawa, Keisuke Yuzu, Naoki Yamamoto, Ken Morishima, Rintaro Inoue, Masaaki Sugiyama, Tetsushi Iwasaki, Masatomo So, Yuji Goto, Atsuo Tamura and Eri Chatani

Molecules 2022, 27(13), 3964; https://doi.org/10.3390/molecules27133964 - 21 Jun 2022

Cited by 2 | Viewed by 1356

Abstract

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously [...] Read more.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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17 pages, 3816 KiB

Open AccessArticle

The Denaturant- and Mutation-Induced Disassembly of Pseudomonas aeruginosa Hexameric Hfq Y55W Mutant

by Victor Marchenkov, Natalia Lekontseva, Natalia Marchenko, Ivan Kashparov, Victoriia Murina, Alexey Nikulin, Vladimir Filimonov and Gennady Semisotnov

Molecules 2022, 27(12), 3821; https://doi.org/10.3390/molecules27123821 - 14 Jun 2022

Cited by 1 | Viewed by 1398

Abstract

Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using [...] Read more.

Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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15 pages, 1678 KiB

Open AccessArticle

Shotgun Proteomics Revealed Preferential Degradation of Misfolded In Vivo Obligate GroE Substrates by Lon Protease in Escherichia coli

by Tatsuya Niwa, Yuhei Chadani and Hideki Taguchi

Molecules 2022, 27(12), 3772; https://doi.org/10.3390/molecules27123772 - 11 Jun 2022

Cited by 2 | Viewed by 1594

Abstract

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend [...] Read more.

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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24 pages, 2842 KiB

Open AccessArticle

CRFalign: A Sequence-Structure Alignment of Proteins Based on a Combination of HMM-HMM Comparison and Conditional Random Fields

by Sung Jong Lee, Keehyoung Joo, Sangjin Sim, Juyong Lee, In-Ho Lee and Jooyoung Lee

Molecules 2022, 27(12), 3711; https://doi.org/10.3390/molecules27123711 - 09 Jun 2022

Cited by 2 | Viewed by 1575

Abstract

Sequence–structure alignment for protein sequences is an important task for the template-based modeling of 3D structures of proteins. Building a reliable sequence–structure alignment is a challenging problem, especially for remote homologue target proteins. We built a method of sequence–structure alignment called CRFalign, which [...] Read more.

Sequence–structure alignment for protein sequences is an important task for the template-based modeling of 3D structures of proteins. Building a reliable sequence–structure alignment is a challenging problem, especially for remote homologue target proteins. We built a method of sequence–structure alignment called CRFalign, which improves upon a base alignment model based on HMM-HMM comparison by employing pairwise conditional random fields in combination with nonlinear scoring functions of structural and sequence features. Nonlinear scoring part is implemented by a set of gradient boosted regression trees. In addition to sequence profile features, various position-dependent structural features are employed including secondary structures and solvent accessibilities. Training is performed on reference alignments at superfamily levels or twilight zone chosen from the SABmark benchmark set. We found that CRFalign method produces relative improvement in terms of average alignment accuracies for validation sets of SABmark benchmark. We also tested CRFalign on 51 sequence–structure pairs involving 15 FM target domains of CASP14, where we could see that CRFalign leads to an improvement in average modeling accuracies in these hard targets (TM-CRFalign

≃ 42.94 %

) compared with that of HHalign (TM-HHalign

≃ 39.05 %

) and also that of MRFalign (TM-MRFalign

≃ 36.93 %

). CRFalign was incorporated to our template search framework called CRFpred and was tested for a random target set of 300 target proteins consisting of Easy, Medium and Hard sets which showed a reasonable template search performance. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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16 pages, 2564 KiB

Open AccessArticle

The Structural Rule Distinguishing a Superfold: A Case Study of Ferredoxin Fold and the Reverse Ferredoxin Fold

by Takumi Nishina, Megumi Nakajima, Masaki Sasai and George Chikenji

Molecules 2022, 27(11), 3547; https://doi.org/10.3390/molecules27113547 - 31 May 2022

Viewed by 1774

Abstract

Superfolds are folds commonly observed among evolutionarily unrelated multiple superfamilies of proteins. Since discovering superfolds almost two decades ago, structural rules distinguishing superfolds from the other ordinary folds have been explored but remained elusive. Here, we analyzed a typical superfold, the ferredoxin fold, [...] Read more.

Superfolds are folds commonly observed among evolutionarily unrelated multiple superfamilies of proteins. Since discovering superfolds almost two decades ago, structural rules distinguishing superfolds from the other ordinary folds have been explored but remained elusive. Here, we analyzed a typical superfold, the ferredoxin fold, and the fold which reverses the N to C terminus direction from the ferredoxin fold as a case study to find the rule to distinguish superfolds from the other folds. Though all the known structural characteristics for superfolds apply to both the ferredoxin fold and the reverse ferredoxin fold, the reverse fold has been found only in a single superfamily. The database analyses in the present study revealed the structural preferences of

α β

- and

β α

-units; the preferences separate two

α

-helices in the ferredoxin fold, preventing their collision and stabilizing the fold. In contrast, in the reverse ferredoxin fold, the preferences bring two helices near each other, inducing structural conflict. The Rosetta folding simulations suggested that the ferredoxin fold is physically much more realizable than the reverse ferredoxin fold. Therefore, we propose that minimal structural conflict or minimal frustration among secondary structures is the rule to distinguish a superfold from ordinary folds. Intriguingly, the database analyses revealed that a most stringent structural rule in proteins, the right-handedness of the

β α β

-unit, is broken in a set of structures to prevent the frustration, suggesting the proposed rule of minimum frustration among secondary structural units is comparably strong as the right-handedness rule of the

β α β

-unit. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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15 pages, 3202 KiB

Open AccessArticle

Advances in Mixer Design and Detection Methods for Kinetics Studies of Macromolecular Folding and Binding on the Microsecond Time Scale

by Takuya Mizukami and Heinrich Roder

Molecules 2022, 27(11), 3392; https://doi.org/10.3390/molecules27113392 - 25 May 2022

Cited by 1 | Viewed by 1424

Abstract

Many important biological processes such as protein folding and ligand binding are too fast to be fully resolved using conventional stopped-flow techniques. Although advances in mixer design and detection methods have provided access to the microsecond time regime, there is room for improvement [...] Read more.

Many important biological processes such as protein folding and ligand binding are too fast to be fully resolved using conventional stopped-flow techniques. Although advances in mixer design and detection methods have provided access to the microsecond time regime, there is room for improvement in terms of temporal resolution and sensitivity. To address this need, we developed a continuous-flow mixing instrument with a dead time of 12 to 27 µs (depending on solution viscosity) and enhanced sensitivity, sufficient for monitoring tryptophan or tyrosine fluorescence changes at fluorophore concentrations as low as 1 µM. Relying on commercially available laser microfabrication services, we obtained an integrated mixer/flow-cell assembly on a quartz chip, based on a cross-channel configuration with channel dimensions and geometry designed to minimize backpressure. By gradually increasing the width of the observation channel downstream from the mixing region, we are able to monitor a reaction progress time window ranging from ~10 µs out to ~3 ms. By combining a solid-state UV laser with a Galvano-mirror scanning strategy, we achieved highly efficient and uniform fluorescence excitation along the flow channel. Examples of applications, including refolding of acid-denatured cytochrome c triggered by a pH jump and binding of a peptide ligand to a PDZ domain, demonstrate the capability of the technique to resolve fluorescence changes down to the 10 µs time regime on modest amounts of reagents. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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20 pages, 3413 KiB

Open AccessArticle

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins

by Max O. D. G. Baker, Nirukshan Shanmugam, Chi L. L. Pham, Sarah R. Ball, Emma Sierecki, Yann Gambin, Megan Steain and Margaret Sunde

Molecules 2022, 27(11), 3382; https://doi.org/10.3390/molecules27113382 - 24 May 2022

Cited by 4 | Viewed by 2736

Abstract

TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation [...] Read more.

TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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14 pages, 3469 KiB

Open AccessArticle

Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3

by Sawaros Onchaiya, Tomonori Saotome, Kenji Mizutani, Jose C. Martinez, Jeremy R. H. Tame, Shun-ichi Kidokoro and Yutaka Kuroda

Molecules 2022, 27(9), 2813; https://doi.org/10.3390/molecules27092813 - 28 Apr 2022

Cited by 2 | Viewed by 1616

Abstract

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ I_n ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported [...] Read more.

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ I_n ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we “reverse engineered” PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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13 pages, 3664 KiB

Open AccessArticle

Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain

by Yunseok Heo, Inhwan Lee, Sunjin Moon, Ji-Hye Yun, Eun Yu Kim, Sam-Yong Park, Jae-Hyun Park, Woo Taek Kim and Weontae Lee

Molecules 2022, 27(7), 2317; https://doi.org/10.3390/molecules27072317 - 02 Apr 2022

Viewed by 2119

Abstract

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the [...] Read more.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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15 pages, 4518 KiB

Open AccessArticle

Effect of Cholesterol Molecules on Aβ1-42 Wild-Type and Mutants Trimers

by Trung Hai Nguyen, Phuong H. Nguyen, Son Tung Ngo and Philippe Derreumaux

Molecules 2022, 27(4), 1395; https://doi.org/10.3390/molecules27041395 - 18 Feb 2022

Cited by 13 | Viewed by 2314

Abstract

Alzheimer’s disease displays aggregates of the amyloid-beta (Aβ) peptide in the brain, and there is increasing evidence that cholesterol may contribute to the pathogenesis of the disease. Though many experimental and theoretical studies have focused on the interactions of Aβ oligomers with membrane [...] Read more.

Alzheimer’s disease displays aggregates of the amyloid-beta (Aβ) peptide in the brain, and there is increasing evidence that cholesterol may contribute to the pathogenesis of the disease. Though many experimental and theoretical studies have focused on the interactions of Aβ oligomers with membrane models containing cholesterol, an understanding of the effect of free cholesterol on small Aβ42 oligomers is not fully established. To address this question, we report on replica exchange with a solute tempering simulation of an Aβ42 trimer with cholesterol and compare it with a previous replica exchange molecular dynamics simulation. We show that the binding hot spots of cholesterol are rather complex, involving hydrophobic residues L17–F20 and L30–M35 with a non-negligible contribution of loop residues D22–K28 and N-terminus residues. We also examine the effects of cholesterol on the trimers of the disease-causing A21G and disease-protective A2T mutations by molecular dynamics simulations. We show that these two mutations moderately impact cholesterol-binding modes. In our REST2 simulations, we find that cholesterol is rarely inserted into aggregates but rather attached as dimers and trimers at the surface of Aβ42 oligomers. We propose that cholesterol acts as a glue to speed up the formation of larger aggregates; this provides a mechanistic link between cholesterol and Alzheimer’s disease. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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14 pages, 4202 KiB

Open AccessArticle

The Pathological G51D Mutation in Alpha-Synuclein Oligomers Confers Distinct Structural Attributes and Cellular Toxicity

by Catherine K. Xu, Marta Castellana-Cruz, Serene W. Chen, Zhen Du, Georg Meisl, Aviad Levin, Benedetta Mannini, Laura S. Itzhaki, Tuomas P. J. Knowles, Christopher M. Dobson, Nunilo Cremades and Janet R. Kumita

Molecules 2022, 27(4), 1293; https://doi.org/10.3390/molecules27041293 - 15 Feb 2022

Cited by 6 | Viewed by 3011

Abstract

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. [...] Read more.

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure–function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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13 pages, 3303 KiB

Open AccessArticle

High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size

by Sara Linse

Molecules 2022, 27(2), 418; https://doi.org/10.3390/molecules27020418 - 10 Jan 2022

Cited by 3 | Viewed by 1539

Abstract

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the [...] Read more.

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein “as is” with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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7 pages, 1401 KiB

Open AccessArticle

Near-Wall Aggregation of Amyloidogenic Aβ 1-40 Peptide: Direct Observation by the FRET

by Natalia Katina, Alisa Mikhaylina, Nelly Ilina, Irina Eliseeva and Vitalii Balobanov

Molecules 2021, 26(24), 7590; https://doi.org/10.3390/molecules26247590 - 15 Dec 2021

Cited by 1 | Viewed by 1784

Abstract

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of [...] Read more.

The formation of amyloid fibrils is one of the variants of the self-organization of polypeptide chains. For the amyloid aggregation, the solution must be oversaturated with proteins. The interface of the liquid (solution) and solid (vessel walls) phases can trigger the adsorption of protein molecules, and the resulting oversaturation can initiate conformational transitions in them. In any laboratory experiment, we cannot exclude the presence of surfaces such as the walls of vessels, cuvettes, etc. However, in many works devoted to the study of amyloid formation, this feature is not considered. In our work, we investigated the behavior of the Aβ 1-40 peptide at the water–glass, water–quartz, and water–plastic interface. We carried out a series of simple experiments and showed that the Aβ 1-40 peptide is actively adsorbed on these surfaces, which leads to a significant interaction and aggregation of peptides. This means that the interface can be the place where the first amyloid nucleus appears. We suggest that this effect may also be one of the reasons for the difficulty of reproducing kinetic data when studying the aggregation of the amyloid of the Aβ 1-40 peptide and other amyloidogenic proteins Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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14 pages, 2206 KiB

Open AccessArticle

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

by Danielle M. Williams, David C. Thorn, Christopher M. Dobson, Sarah Meehan, Sophie E. Jackson, Joanna M. Woodcock and John A. Carver

Molecules 2021, 26(20), 6120; https://doi.org/10.3390/molecules26206120 - 11 Oct 2021

Cited by 8 | Viewed by 2530

Abstract

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated [...] Read more.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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Review

Jump to: Research

10 pages, 1895 KiB

Open AccessReview

Conformational Variability of Amyloid-β and the Morphological Diversity of Its Aggregates

by Maho Yagi-Utsumi and Koichi Kato

Molecules 2022, 27(15), 4787; https://doi.org/10.3390/molecules27154787 - 26 Jul 2022

Cited by 6 | Viewed by 1699

Abstract

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many [...] Read more.

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-β (Aβ) characterized from experimental and theoretical approaches. Aβ exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the β-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aβ amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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16 pages, 3478 KiB

Open AccessReview

Supersaturation-Dependent Formation of Amyloid Fibrils

by Yuji Goto, Masahiro Noji, Kichitaro Nakajima and Keiichi Yamaguchi

Molecules 2022, 27(14), 4588; https://doi.org/10.3390/molecules27144588 - 19 Jul 2022

Cited by 5 | Viewed by 2477

Abstract

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute’s concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases [...] Read more.

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute’s concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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21 pages, 2361 KiB

Open AccessReview

The Wako-Saitô-Muñoz-Eaton Model for Predicting Protein Folding and Dynamics

by Koji Ooka, Runjing Liu and Munehito Arai

Molecules 2022, 27(14), 4460; https://doi.org/10.3390/molecules27144460 - 12 Jul 2022

Cited by 6 | Viewed by 2761

Abstract

Despite the recent advances in the prediction of protein structures by deep neutral networks, the elucidation of protein-folding mechanisms remains challenging. A promising theory for describing protein folding is a coarse-grained statistical mechanical model called the Wako-Saitô-Muñoz-Eaton (WSME) model. The model can calculate [...] Read more.

Despite the recent advances in the prediction of protein structures by deep neutral networks, the elucidation of protein-folding mechanisms remains challenging. A promising theory for describing protein folding is a coarse-grained statistical mechanical model called the Wako-Saitô-Muñoz-Eaton (WSME) model. The model can calculate the free-energy landscapes of proteins based on a three-dimensional structure with low computational complexity, thereby providing a comprehensive understanding of the folding pathways and the structure and stability of the intermediates and transition states involved in the folding reaction. In this review, we summarize previous and recent studies on protein folding and dynamics performed using the WSME model and discuss future challenges and prospects. The WSME model successfully predicted the folding mechanisms of small single-domain proteins and the effects of amino-acid substitutions on protein stability and folding in a manner that was consistent with experimental results. Furthermore, extended versions of the WSME model were applied to predict the folding mechanisms of multi-domain proteins and the conformational changes associated with protein function. Thus, the WSME model may contribute significantly to solving the protein-folding problem and is expected to be useful for predicting protein folding, stability, and dynamics in basic research and in industrial and medical applications. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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12 pages, 303 KiB

Open AccessReview

The Molten Globule State of a Globular Protein in a Cell Is More or Less Frequent Case Rather than an Exception

by Valentina E. Bychkova, Dmitry A. Dolgikh, Vitalii A. Balobanov and Alexei V. Finkelstein

Molecules 2022, 27(14), 4361; https://doi.org/10.3390/molecules27144361 - 07 Jul 2022

Cited by 4 | Viewed by 1735

Abstract

Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its [...] Read more.

Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn’s hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

23 pages, 4315 KiB

Open AccessReview

Functional Bacterial Amyloids: Understanding Fibrillation, Regulating Biofilm Fibril Formation and Organizing Surface Assemblies

by Thorbjørn Vincent Sønderby, Zahra Najarzadeh and Daniel Erik Otzen

Molecules 2022, 27(13), 4080; https://doi.org/10.3390/molecules27134080 - 24 Jun 2022

Cited by 15 | Viewed by 3188

Abstract

Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA (E. coli) and FapC (Pseudomonas) are assembled as functional bacterial amyloid (FuBA) on the cell surface in a carefully optimized [...] Read more.

Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA (E. coli) and FapC (Pseudomonas) are assembled as functional bacterial amyloid (FuBA) on the cell surface in a carefully optimized process. Besides a host of helper proteins, FuBA formation is aided by multiple imperfect repeats which stabilize amyloid and streamline the aggregation mechanism to a fast-track assembly dominated by primary nucleation. These repeats, which are found in variable numbers in Pseudomonas, are most likely the structural core of the fibrils, though we still lack experimental data to determine whether the repeats give rise to β-helix structures via stacked β-hairpins (highly likely for CsgA) or more complicated arrangements (possibly the case for FapC). The response of FuBA fibrillation to denaturants suggests that nucleation and elongation involve equal amounts of folding, but protein chaperones preferentially target nucleation for effective inhibition. Smart peptides can be designed based on these imperfect repeats and modified with various flanking sequences to divert aggregation to less stable structures, leading to a reduction in biofilm formation. Small molecules such as EGCG can also divert FuBA to less organized structures, such as partially-folded oligomeric species, with the same detrimental effect on biofilm. Finally, the strong tendency of FuBA to self-assemble can lead to the formation of very regular two-dimensional amyloid films on structured surfaces such as graphite, which strongly implies future use in biosensors or other nanobiomaterials. In summary, the properties of functional amyloid are a much-needed corrective to the unfortunate association of amyloid with neurodegenerative disease and a testimony to nature’s ability to get the best out of a protein fold. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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17 pages, 2158 KiB

Open AccessReview

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science

by Kunihiro Kuwajima, Maho Yagi-Utsumi, Saeko Yanaka and Koichi Kato

Molecules 2022, 27(12), 3748; https://doi.org/10.3390/molecules27123748 - 10 Jun 2022

Cited by 4 | Viewed by 2179

Abstract

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching [...] Read more.

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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29 pages, 8888 KiB

Open AccessReview

Molecular Dynamics Simulation Studies on the Aggregation of Amyloid-β Peptides and Their Disaggregation by Ultrasonic Wave and Infrared Laser Irradiation

by Hisashi Okumura and Satoru G. Itoh

Molecules 2022, 27(8), 2483; https://doi.org/10.3390/molecules27082483 - 12 Apr 2022

Cited by 8 | Viewed by 3359

Abstract

Alzheimer’s disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-β (Aβ) peptides. This review article presents molecular dynamics (MD) simulation studies of Aβ peptides and Aβ fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, [...] Read more.

Alzheimer’s disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-β (Aβ) peptides. This review article presents molecular dynamics (MD) simulation studies of Aβ peptides and Aβ fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, and disruption of the amyloid fibril by ultrasonic wave and infrared laser irradiation. In the aggregation of Aβ, a β-hairpin structure promotes the formation of intermolecular β-sheet structures. Aβ peptides tend to exist at hydrophilic/hydrophobic interfaces and form more β-hairpin structures than in bulk water. These facts are the reasons why the aggregation is accelerated at the interface. We also explain how polyphenols, which are attracting attention as aggregation inhibitors of Aβ peptides, interact with Aβ. An MD simulation study of the Aβ amyloid fibrils in equilibrium is also presented: the Aβ amyloid fibril has a different structure at one end from that at the other end. The amyloid fibrils can be destroyed by ultrasonic wave and infrared laser irradiation. The molecular mechanisms of these amyloid fibril disruptions are also explained, particularly focusing on the function of water molecules. Finally, we discuss the prospects for developing treatments for Alzheimer’s disease using MD simulations. Full article

(This article belongs to the Special Issue Frontiers in Protein Folding and Related Areas – in Memory of Professor Sir Christopher M. Dobson (1949–2019))

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