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Biomolecules, Volume 3, Issue 4 (December 2013), Pages 733-1052

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Research

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Open AccessArticle Isolation, NMR Spectral Analysis and Hydrolysis Studies of a Hepta Pyranosyl Diterpene Glycoside from Stevia rebaudiana Bertoni
Biomolecules 2013, 3(4), 733-740; doi:10.3390/biom3040733
Received: 29 August 2013 / Revised: 18 September 2013 / Accepted: 18 September 2013 / Published: 30 September 2013
Cited by 1 | PDF Full-text (396 KB) | HTML Full-text | XML Full-text
Abstract
From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a minor steviol glycoside, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-(3-O-β-D-glucopyranosyl-α-L-rhamnopyranosyl)-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1); also known as rebaudioside O having seven sugar units has
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From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a minor steviol glycoside, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-(3-O-β-D-glucopyranosyl-α-L-rhamnopyranosyl)-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1); also known as rebaudioside O having seven sugar units has been isolated. Its structural characterization has been achieved by the extensive 1D (1H and 13C), and 2D NMR (COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside O using acid and enzymatic methods to identify aglycone and sugar residues in its structure as well as their configurations. Full article
Open AccessArticle Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase
Biomolecules 2013, 3(4), 848-869; doi:10.3390/biom3040848
Received: 7 August 2013 / Revised: 27 September 2013 / Accepted: 12 October 2013 / Published: 21 October 2013
Cited by 2 | PDF Full-text (949 KB) | HTML Full-text | XML Full-text
Abstract
Protein disulfide isomerases (PDIs) catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER). Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′) and two non-catalytic domains (
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Protein disulfide isomerases (PDIs) catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER). Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′) and two non-catalytic domains (b, b′), in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6). Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11) possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP) fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6) functionally complemented dsbA. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER. Full article
(This article belongs to the Special Issue Protein Folding and Misfolding)
Open AccessArticle Structural Evidence for the Tetrameric Assembly of Chemokine CCL11 and the Glycosaminoglycan Arixtra™
Biomolecules 2013, 3(4), 905-922; doi:10.3390/biom3040905
Received: 22 August 2013 / Revised: 23 October 2013 / Accepted: 29 October 2013 / Published: 6 November 2013
Cited by 2 | PDF Full-text (778 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native
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Understanding chemokine interactions with glycosaminoglycans (GAG) is critical as these interactions have been linked to a number of inflammatory medical conditions, such as arthritis and asthma. To better characterize in vivo protein function, comprehensive knowledge of multimeric species, formed by chemokines under native conditions, is necessary. Herein is the first report of a tetrameric assembly of the human chemokine CCL11, which was shown bound to the GAG Arixtra™. Isothermal titration calorimetry data indicated that CCL11 interacts with Arixtra, and ion mobility mass spectrometry (IM-MS) was used to identify ions corresponding to the CCL11 tetrameric species bound to Arixtra. Collisional cross sections (CCS) of the CCL11 tetramer-Arixtra noncovalent complex were compared to theoretical CCS values calculated using a preliminary structure of the complex deduced using X-ray crystallography. Experimental CCS values were in agreement with theoretical values, strengthening the IM-MS evidence for the formation of the noncovalent complex. Tandem mass spectrometry data of the complex indicated that the tetramer-GAG complex dissociates into a monomer and a trimer-GAG species, suggesting that two CC-like dimers are bridged by Arixtra. As development of chemokine inhibitors is of utmost importance to treatment of medical inflammatory conditions, these results provide vital insights into chemokine-GAG interactions. Full article
(This article belongs to the Special Issue Focus Update in Biomolecules)
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Open AccessArticle Unfolding Thermodynamics of Cysteine-Rich Proteins and Molecular Thermal-Adaptation of Marine Ciliates
Biomolecules 2013, 3(4), 967-985; doi:10.3390/biom3040967
Received: 9 September 2013 / Revised: 28 October 2013 / Accepted: 29 October 2013 / Published: 18 November 2013
Cited by 4 | PDF Full-text (961 KB) | HTML Full-text | XML Full-text
Abstract
Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative
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Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones. Full article
(This article belongs to the Special Issue Protein Folding and Misfolding)
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Open AccessArticle Control of Collagen Stability and Heterotrimer Specificity through Repulsive Electrostatic Interactions
Biomolecules 2013, 3(4), 986-996; doi:10.3390/biom3040986
Received: 29 October 2013 / Revised: 27 November 2013 / Accepted: 28 November 2013 / Published: 6 December 2013
Cited by 4 | PDF Full-text (1336 KB) | HTML Full-text | XML Full-text
Abstract
Charge-pair interactions between acidic and basic residues on the surface of collagen can promote stability as well as control specificity of molecular recognition. Heterotrimeric collagen peptides have been engineered de novo using either rational or computational methods, which in both cases optimize networks
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Charge-pair interactions between acidic and basic residues on the surface of collagen can promote stability as well as control specificity of molecular recognition. Heterotrimeric collagen peptides have been engineered de novo using either rational or computational methods, which in both cases optimize networks of favorable charge-pair interactions in the target structure. Less understood is the role of electrostatic repulsion between groups of like charge in destabilizing structure or directing molecular recognition. To study this, we apply a “charge crowding” approach, where repulsive interactions between multiple aspartate side chains are found to destabilize the homotrimer states in triple helical peptide system and can be utilized to promote the formation of heterotrimers. Neutralizing surface charge by increasing salt concentration or decreasing pH can enhance homotrimer stability, confirming the role of charge crowding on the destabilization of homotrimers via electrostatic repulsion. Charge crowding may be used in conjunction with other approaches to create specific collagen heterotrimers. Full article
(This article belongs to the Special Issue Protein Folding and Misfolding)
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Review

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Open AccessReview Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals
Biomolecules 2013, 3(4), 741-777; doi:10.3390/biom3040741
Received: 23 August 2013 / Revised: 22 September 2013 / Accepted: 23 September 2013 / Published: 2 October 2013
Cited by 21 | PDF Full-text (3082 KB) | HTML Full-text | XML Full-text
Abstract
Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous
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Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived there from for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral alcohols and unntural aminoacids for pharmaceuticals. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications) Print Edition available
Open AccessReview Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis
Biomolecules 2013, 3(4), 778-811; doi:10.3390/biom3040778
Received: 21 August 2013 / Revised: 22 September 2013 / Accepted: 23 September 2013 / Published: 8 October 2013
Cited by 4 | PDF Full-text (1286 KB) | HTML Full-text | XML Full-text
Abstract
Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance
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Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications) Print Edition available
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Open AccessReview Biocatalysis for Biobased Chemicals
Biomolecules 2013, 3(4), 812-847; doi:10.3390/biom3040812
Received: 3 September 2013 / Revised: 8 October 2013 / Accepted: 8 October 2013 / Published: 17 October 2013
Cited by 13 | PDF Full-text (674 KB) | HTML Full-text | XML Full-text
Abstract
The design and development of greener processes that are safe and friendly is an irreversible trend that is driven by sustainable and economic issues. The use of Biocatalysis as part of a manufacturing process fits well in this trend as enzymes are themselves
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The design and development of greener processes that are safe and friendly is an irreversible trend that is driven by sustainable and economic issues. The use of Biocatalysis as part of a manufacturing process fits well in this trend as enzymes are themselves biodegradable, require mild conditions to work and are highly specific and well suited to carry out complex reactions in a simple way. The growth of computational capabilities in the last decades has allowed Biocatalysis to develop sophisticated tools to understand better enzymatic phenomena and to have the power to control not only process conditions but also the enzyme’s own nature. Nowadays, Biocatalysis is behind some important products in the pharmaceutical, cosmetic, food and bulk chemicals industry. In this review we want to present some of the most representative examples of industrial chemicals produced in vitro through enzymatic catalysis. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications) Print Edition available
Open AccessReview Biotechnological Applications of Transglutaminases
Biomolecules 2013, 3(4), 870-888; doi:10.3390/biom3040870
Received: 12 September 2013 / Revised: 10 October 2013 / Accepted: 11 October 2013 / Published: 22 October 2013
Cited by 12 | PDF Full-text (764 KB) | HTML Full-text | XML Full-text
Abstract
In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather.
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In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather. In recent years, biotechnological applications of transglutaminases have come to light in areas ranging from material sciences to medicine. There has also been a substantial effort to further investigate the fundamentals of transglutaminases, as many of their characteristics that remain poorly understood. Those studies also work towards the goal of developing transglutaminases as more efficient catalysts. Progress in this area includes structural information and novel chemical and biological assays. Here, we review recent achievements in this area in order to illustrate the versatility of transglutaminases. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications) Print Edition available
Open AccessReview Biophysical Insights into the Inhibitory Mechanism of Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors
Biomolecules 2013, 3(4), 889-904; doi:10.3390/biom3040889
Received: 18 September 2013 / Revised: 22 October 2013 / Accepted: 22 October 2013 / Published: 1 November 2013
PDF Full-text (425 KB) | HTML Full-text | XML Full-text
Abstract
HIV-1 reverse transcriptase (RT) plays a central role in HIV infection. Current United States Federal Drug Administration (USFDA)-approved antiretroviral therapies can include one of five approved non-nucleoside RT inhibitors (NNRTIs), which are potent inhibitors of RT activity. Despite their crucial clinical role in
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HIV-1 reverse transcriptase (RT) plays a central role in HIV infection. Current United States Federal Drug Administration (USFDA)-approved antiretroviral therapies can include one of five approved non-nucleoside RT inhibitors (NNRTIs), which are potent inhibitors of RT activity. Despite their crucial clinical role in treating and preventing HIV-1 infection, their mechanism of action remains elusive. In this review, we introduce RT and highlight major advances from experimental and computational biophysical experiments toward an understanding of RT function and the inhibitory mechanism(s) of NNRTIs. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications) Print Edition available
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Open AccessReview Research Applications of Proteolytic Enzymes in Molecular Biology
Biomolecules 2013, 3(4), 923-942; doi:10.3390/biom3040923
Received: 15 October 2013 / Revised: 4 November 2013 / Accepted: 6 November 2013 / Published: 8 November 2013
Cited by 5 | PDF Full-text (659 KB) | HTML Full-text | XML Full-text
Abstract
Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role
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Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications) Print Edition available
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Open AccessReview Control of Cell Differentiation by Mitochondria, Typically Evidenced in Dictyostelium Development
Biomolecules 2013, 3(4), 943-966; doi:10.3390/biom3040943
Received: 28 September 2013 / Revised: 1 November 2013 / Accepted: 2 November 2013 / Published: 11 November 2013
Cited by 7 | PDF Full-text (1075 KB) | HTML Full-text | XML Full-text
Abstract
In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP) synthesis by respiration. Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination,
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In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP) synthesis by respiration. Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination, cell movement, and pattern formation. This has been most strikingly realized in development of the cellular slime mold Dictyostelium. For example, the expression of the mitochondrial ribosomal protein S4 (mt-rps4) gene is required for the initial differentiation. The Dictyostelium homologue (Dd-TRAP1) of TRAP-1 (tumor necrosis receptor-associated protein 1), a mitochondrial molecular chaperone belonging to the Hsp90 family, allows the prompt transition of cells from growth to differentiation through a novel prestarvation factor (PSF-3) in growth medium. Moreover, a cell-type-specific organelle named a prespore-specific vacuole (PSV) is constructed by mitochondrial transformation with the help of the Golgi complex. Mitochondria are also closely involved in a variety of cellular activities including CN-resistant respiration and apoptosis. These mitochondrial functions are reviewed in this article, with special emphasis on the regulation of Dictyostelium development. Full article
(This article belongs to the Special Issue Focus Update in Biomolecules)
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Open AccessReview Toxin Instability and Its Role in Toxin Translocation from the Endoplasmic Reticulum to the Cytosol
Biomolecules 2013, 3(4), 997-1029; doi:10.3390/biom3040997
Received: 4 November 2013 / Revised: 26 November 2013 / Accepted: 27 November 2013 / Published: 10 December 2013
Cited by 3 | PDF Full-text (501 KB) | HTML Full-text | XML Full-text
Abstract
AB toxins enter a host cell by receptor-mediated endocytosis. The catalytic A chain then crosses the endosome or endoplasmic reticulum (ER) membrane to reach its cytosolic target. Dissociation of the A chain from the cell-binding B chain occurs before or during translocation to
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AB toxins enter a host cell by receptor-mediated endocytosis. The catalytic A chain then crosses the endosome or endoplasmic reticulum (ER) membrane to reach its cytosolic target. Dissociation of the A chain from the cell-binding B chain occurs before or during translocation to the cytosol, and only the A chain enters the cytosol. In some cases, AB subunit dissociation is facilitated by the unique physiology and function of the ER. The A chains of these ER-translocating toxins are stable within the architecture of the AB holotoxin, but toxin disassembly results in spontaneous or assisted unfolding of the isolated A chain. This unfolding event places the A chain in a translocation-competent conformation that promotes its export to the cytosol through the quality control mechanism of ER-associated degradation. A lack of lysine residues for ubiquitin conjugation protects the exported A chain from degradation by the ubiquitin-proteasome system, and an interaction with host factors allows the cytosolic toxin to regain a folded, active state. The intrinsic instability of the toxin A chain thus influences multiple steps of the intoxication process. This review will focus on the host–toxin interactions involved with A chain unfolding in the ER and A chain refolding in the cytosol. Full article
(This article belongs to the Special Issue Protein Folding and Misfolding)
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Open AccessReview Protein Stability, Folding and Misfolding in Human PGK1 Deficiency
Biomolecules 2013, 3(4), 1030-1052; doi:10.3390/biom3041030
Received: 21 October 2013 / Revised: 6 December 2013 / Accepted: 13 December 2013 / Published: 18 December 2013
Cited by 4 | PDF Full-text (873 KB) | HTML Full-text | XML Full-text
Abstract
Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein
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Conformational diseases are often caused by mutations, altering protein folding and stability in vivo. We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients. Full article
(This article belongs to the Special Issue Protein Folding and Misfolding)
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