Biomolecules

Editorial

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2 pages, 178 KiB

Open AccessEditorial

Editorial for the Special Issue: “State-of-Art in Protein Engineering”

by Lada E. Petrovskaya and Dmitry A. Dolgikh

Biomolecules 2022, 12(7), 966; https://doi.org/10.3390/biom12070966 - 10 Jul 2022

Viewed by 1331

Abstract

This Special Issue of Biomolecules demonstrates the almost unlimited possibilities of modern protein engineering in gene expression, protein production and modification, as well as the design and creation of new proteins [...] Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

Research

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15 pages, 2519 KiB

Open AccessArticle

Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c

by Rita V. Chertkova, Alexander M. Firsov, Nadezda A. Brazhe, Evelina I. Nikelshparg, Zhanna V. Bochkova, Tatyana V. Bryantseva, Marina A. Semenova, Adil A. Baizhumanov, Elena A. Kotova, Mikhail P. Kirpichnikov, Georgy V. Maksimov, Yuriy N. Antonenko and Dmitry A. Dolgikh

Biomolecules 2022, 12(5), 665; https://doi.org/10.3390/biom12050665 - 04 May 2022

Cited by 8 | Viewed by 2001

Abstract

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial [...] Read more.

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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12 pages, 2324 KiB

Open AccessEditor’s ChoiceArticle

Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates

by Yuehui Tian, Shang Yang, Georg Nagel and Shiqiang Gao

Biomolecules 2022, 12(1), 88; https://doi.org/10.3390/biom12010088 - 06 Jan 2022

Cited by 3 | Viewed by 3114

Abstract

Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs [...] Read more.

Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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22 pages, 7573 KiB

Open AccessArticle

Bispecific Antibodies for IFN-β Delivery to ErbB2⁺ Tumors

by Vladislav S. Rybchenko, Anna A. Panina, Teimur K. Aliev, Olga N. Solopova, Dmitry S. Balabashin, Valery N. Novoseletsky, Dmitry A. Dolgikh, Petr G. Sveshnikov and Mikhail P. Kirpichnikov

Biomolecules 2021, 11(12), 1915; https://doi.org/10.3390/biom11121915 - 20 Dec 2021

Cited by 4 | Viewed by 2818

Abstract

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2⁺ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is [...] Read more.

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2⁺ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2⁺ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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14 pages, 7460 KiB

Open AccessArticle

Identification and Biochemical Characterization of a Novel Hormone-Sensitive Lipase Family Esterase Est19 from the Antarctic Bacterium Pseudomonas sp. E2-15

by Xiaoyu Liu, Mingyang Zhou, Shu Xing, Tao Wu, Hailun He, John Kevin Bielicki and Jianbin Chen

Biomolecules 2021, 11(11), 1552; https://doi.org/10.3390/biom11111552 - 20 Oct 2021

Cited by 12 | Viewed by 2024

Abstract

Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active [...] Read more.

Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser¹⁵⁵, Asp²⁵³, and His²⁸² residues. The catalytic efficiency (k_cat/K_m) of Est19 for the pNPC6 substrate is 148.68 s⁻¹mM⁻¹ at 40 °C. Replacing Glu¹⁵⁴ juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10–60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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13 pages, 3170 KiB

Open AccessArticle

Design and Prototyping of Genetically Encoded Arsenic Biosensors Based on Transcriptional Regulator AfArsR

by Salma Saeed Khan, Yi Shen, Muhammad Qaiser Fatmi, Robert E. Campbell and Habib Bokhari

Biomolecules 2021, 11(9), 1276; https://doi.org/10.3390/biom11091276 - 26 Aug 2021

Cited by 6 | Viewed by 2392

Abstract

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As³⁺) is highly toxic to cells due to its ability [...] Read more.

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As³⁺) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disruption of protein–protein interactions, and eventually to cell death. A genetically encoded biosensor for the detection of As³⁺ could potentially facilitate the investigation of such toxicity both in vitro and in vivo. Here, we designed and developed two prototype genetically encoded arsenic biosensors (GEARs), based on a bacterial As³⁺ responsive transcriptional factor AfArsR from Acidithiobacillus ferrooxidans. We constructed FRET-based GEAR biosensors by insertion of AfArsR between FP acceptor/donor FRET pairs. We further designed and engineered single FP-based GEAR biosensors by insertion of AfArsR into GFP. These constructs represent prototypes for a new family of biosensors based on the ArsR transcriptional factor scaffold. Further improvements of the GEAR biosensor family could lead to variants with suitable performance for detection of As³⁺ in various biological and environmental systems. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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16 pages, 3275 KiB

Open AccessArticle

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content

by Yana Y. Berlina, Lada E. Petrovskaya, Elena A. Kryukova, Lyudmila N. Shingarova, Sultan Sh. Gapizov, Mariya V. Kryukova, Elizaveta M. Rivkina, Mikhail P. Kirpichnikov and Dmitry A. Dolgikh

Biomolecules 2021, 11(8), 1229; https://doi.org/10.3390/biom11081229 - 17 Aug 2021

Cited by 9 | Viewed by 2298

Abstract

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features [...] Read more.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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Review

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12 pages, 1970 KiB

Open AccessReview

GroEL—A Versatile Chaperone for Engineering and a Plethora of Applications

by Maria S. Yurkova and Alexey N. Fedorov

Biomolecules 2022, 12(5), 607; https://doi.org/10.3390/biom12050607 - 19 Apr 2022

Cited by 8 | Viewed by 2289

Abstract

Chaperones play a vital role in the life of cells by facilitating the correct folding of other proteins and maintaining them in a functional state, being themselves, as a rule, more stable than the rest of cell proteins. Their functional properties naturally tempt [...] Read more.

Chaperones play a vital role in the life of cells by facilitating the correct folding of other proteins and maintaining them in a functional state, being themselves, as a rule, more stable than the rest of cell proteins. Their functional properties naturally tempt investigators to actively adapt them for biotechnology needs. This review will mostly focus on the applications found for the bacterial chaperonin GroE and its counterparts from other organisms, in biotechnology or for research purposes, both in their engineered or intact versions. Full article

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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