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Special Issue "Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday"

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A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Biomacromolecules: Proteins".

Deadline for manuscript submissions: closed (15 April 2023) | Viewed by 50454

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Special Issue Editors

Prof. Dr. Prakash Kulkarni

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Guest Editor

Department of Medical Oncology, City of Hope National Medical Center, Duarte, CA 91010, USA
Interests: cancer biology; prostate cancer; solid tumors
Special Issues, Collections and Topics in MDPI journals

Dr. Stefania Brocca

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Department of Biotechnology and Biosciences, University of Milan-Bicocca, Milan, Italy
Interests: intrinsically disordered proteins; phase separations; charge-induced conformational transitions; structural and functional properties of hydrolytic enzymes
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Keith Dunker
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Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA
Interests: intrinsically disordered proteins
Special Issues, Collections and Topics in MDPI journals

Dr. Sonia Longhi

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Architecture and Function of Biological Macromolecules (AFMB), UMR 7257 CNRS & Aix-Marseille University, 13288 Marseille, France
Interests: intrinsically disordered proteins; folding copuled to binding; protein-protein interactions; structural transitions; paramyxoviruses
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Professor Vladimir Uversky

The discovery of intrinsically disordered proteins (IDPs) has changed our perception of proteins as existing either in their native, folded and, hence, active state or as denatured, unfolded, and non-functional state. We now know that proteins may exist as dynamic conformational ensembles with little or no secondary and tertiary structure and yet be functional. Although apparently posing a challenge to Anfinsen’s postulate, IDPs in fact represent the limits of the structure–function paradigm.

IDPs also pose an additional challenge. Structural disorder is not easily tractable/detectable experimentally using techniques and approaches traditionally used by structuralists, and these techniques need to be enriched and complemented by those typically used by polymer physicists and chemists. Indeed, for the same reason, IDPs, which are prevalent in all three kingdoms of life and comprise almost half the human proteome, are often referred to as constituents of the ‘dark’ matter of biology. Over the past almost thirty years, studies on IDPs have fuelled crosstalk between biochemists, structuralists, and molecular biologists on one hand, and physicists and chemists on the other. These different communities have therefore faced the need to understand each other while speaking different languages.

Prof. Vladimir Uversky, one of the pioneers in the IDP field, realised the necessity of this dialogue and published an eloquent and thought-provoking paper, ‘Natively unfolded proteins: a point where biology waits for physics’ (Protein Sci. 2002). Today, twenty years later, to stimulate reflections on this issue and ponder the role of physical sciences in unravelling the structure–function paradigm, Biomolecules will be publishing this Special Issue on the occasion of Prof. Uversky’s 60th birthday celebrating his many contributions to the IDP field. This Special Issue aims to present the state of the art and invites contributions from those researchers who have known Prof. Vladimir Uversky personally.

Prof. Dr. Prakash Kulkarni
Dr. Stefania Brocca
Prof. Dr. Keith Dunker
Dr. Sonia Longhi
Guest Editors

Manuscript Submission Information

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

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biomolecules is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

intrinsically disordered proteins (IDPs)
intrinsically disordered regions (IDRs)
intrinsic disorder prediction
intrinsic disorder characterization & methodological developments
molecular recognition & regulation mediated by structural disorder
conformational ensembles
energy landscape of IDPs
modeling of IDPs
conformational and binding properties of IDPs
folding upon binding
liquid-liquid phase separation
fibrillation
IDPs in nanotechnoogy and biotechnology
intrinsic disorder in disease and evolution

Published Papers (26 papers)

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Editorial

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8 pages, 793 KiB

Open AccessEditorial

Per Aspera ad Chaos: Vladimir Uversky’s Odyssey through the Strange World of Intrinsically Disordered Proteins

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Biomolecules 2023, 13(6), 1015; https://doi.org/10.3390/biom13061015 - 19 Jun 2023

Viewed by 1036

Abstract

Until the late 1990s, we believed that protein function required a unique, well-defined 3D structure encrypted in the amino acid sequence [...] Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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Research

Jump to: Editorial, Review

17 pages, 2645 KiB

Open AccessArticle

Effects of Mass Change on Liquid–Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma

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Biomolecules 2023, 13(4), 625; https://doi.org/10.3390/biom13040625 - 30 Mar 2023

Cited by 1 | Viewed by 1010

Abstract

In recent years, many experimental and theoretical studies of protein liquid–liquid phase separation (LLPS) have shown its important role in the processes of physiology and pathology. However, there is a lack of definite information on the regulation mechanism of LLPS in vital activities. Recently, we found that the intrinsically disordered proteins with the insertion/deletion of a non-interacting peptide segment or upon isotope replacement could form droplets, and the LLPS states are different from the proteins without those. We believed that there is an opportunity to decipher the LLPS mechanism with the mass change perspective. To investigate the effect of molecular mass on LLPS, we developed a coarse-grained model with different bead masses, including mass 1.0, mass 1.1, mass 1.2, mass 1.3, and mass 1.5 in atomic units or with the insertion of a non-interacting peptide (10 aa) and performed molecular dynamic simulations. Consequently, we found that the mass increase promotes the LLPS stability, which is based on decreasing the z motion rate and increasing the density and the inter-chain interaction of droplets. This insight into LLPS by mass change paves the way for the regulation and relevant diseases on LLPS. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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17 pages, 2998 KiB

Open AccessArticle

Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ

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Biomolecules 2023, 13(3), 404; https://doi.org/10.3390/biom13030404 - 21 Feb 2023

Cited by 1 | Viewed by 1186

Abstract

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2–4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2–4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2–4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2–4, with long and short linkers, forms heterogeneous complexes, whereas QT4–6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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10 pages, 2243 KiB

Open AccessArticle

Conformational Analysis of Charged Homo-Polypeptides

Lavi S. Bigman

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Yaakov Levy

Biomolecules 2023, 13(2), 363; https://doi.org/10.3390/biom13020363 - 15 Feb 2023

Cited by 1 | Viewed by 1113

Abstract

Many proteins have intrinsically disordered regions (IDRs), which are often characterized by a high fraction of charged residues with polyampholytic (i.e., mixed charge) or polyelectrolytic (i.e., uniform charge) characteristics. Polyelectrolytic IDRs include consecutive positively charged Lys or Arg residues (K/R repeats) or consecutive negatively charged Asp or Glu residues (D/E repeats). In previous research, D/E repeats were found to be about five times longer than K/R repeats and to be much more common in eukaryotes. Within these repeats, a preference is often observed for E over D and for K over R. To understand the greater prevalence of D/E over K/R repeats and the higher abundance of E and K, we simulated the conformational ensemble of charged homo-polypeptides (polyK, polyR, polyD, and polyE) using molecular dynamics simulations. The conformational preferences and dynamics of these polyelectrolytic polypeptides change with changes in salt concentration. In particular, polyD and polyE are more sensitive to salt than polyK and polyR, as polyD and polyE tend to adsorb more divalent cations, which leads to their having more compact conformations. We conclude with a discussion of biophysical explanations for the relative abundance of charged amino acids and particularly for the greater abundance of D/E repeats over K/R repeats. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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17 pages, 1759 KiB

Open AccessArticle

Different Forms of Disorder in NMDA-Sensitive Glutamate Receptor Cytoplasmic Domains Are Associated with Differences in Condensate Formation

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Biomolecules 2023, 13(1), 4; https://doi.org/10.3390/biom13010004 - 20 Dec 2022

Cited by 4 | Viewed by 1438

Abstract

The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDAR) helps assemble downstream signaling pathways through protein interactions within the postsynaptic density (PSD), which are mediated by its intracellular C-terminal domain (CTD). The most abundant NMDAR subunits in the brain are GluN2A and GluN2B, which are associated with a developmental switch in NMDAR composition. Previously, we used single molecule fluorescence resonance energy transfer (smFRET) to show that the GluN2B CTD contained an intrinsically disordered region with slow, hop-like conformational dynamics. The CTD from GluN2B also undergoes liquid–liquid phase separation (LLPS) with synaptic proteins. Here, we extend these observations to the GluN2A CTD. Sequence analysis showed that both subunits contain a form of intrinsic disorder classified as weak polyampholytes. However, only GluN2B contained matched patterning of arginine and aromatic residues, which are linked to LLPS. To examine the conformational distribution, we used discrete molecular dynamics (DMD), which revealed that GluN2A favors extended disordered states containing secondary structures while GluN2B favors disordered globular states. In contrast to GluN2B, smFRET measurements found that GluN2A lacked slow conformational dynamics. Thus, simulation and experiments found differences in the form of disorder. To understand how this affects protein interactions, we compared the ability of these two NMDAR isoforms to undergo LLPS. We found that GluN2B readily formed condensates with PSD-95 and SynGAP, while GluN2A failed to support LLPS and instead showed a propensity for colloidal aggregation. That GluN2A fails to support this same condensate formation suggests a developmental switch in LLPS propensity. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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15 pages, 1947 KiB

Open AccessArticle

Portability of a Small-Molecule Binding Site between Disordered Proteins

Rajesh Jaiprashad

Sachith Roch De Silva

Lisette M. Fred Lucena

Ella Meyer

and

Steven J. Metallo

Biomolecules 2022, 12(12), 1887; https://doi.org/10.3390/biom12121887 - 16 Dec 2022

Cited by 1 | Viewed by 1482

Abstract

Intrinsically disordered proteins (IDPs) are important in both normal and disease states. Small molecules can be targeted to disordered regions, but we currently have only a limited understanding of the nature of small-molecule binding sites in IDPs. Here, we show that a minimal small-molecule binding sequence of eight contiguous residues derived from the Myc protein can be ported into a different disordered protein and recapitulate small-molecule binding activity in the new context. We also find that the residue immediately flanking the binding site can have opposing effects on small-molecule binding in the different disordered protein contexts. The results demonstrate that small-molecule binding sites can act modularly and are portable between disordered protein contexts but that residues outside of the minimal binding site can modulate binding affinity. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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25 pages, 2172 KiB

Open AccessArticle

The Role of Membrane Affinity and Binding Modes in Alpha-Synuclein Regulation of Vesicle Release and Trafficking

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Biomolecules 2022, 12(12), 1816; https://doi.org/10.3390/biom12121816 - 05 Dec 2022

Cited by 4 | Viewed by 1539

Abstract

Alpha-synuclein is a presynaptic protein linked to Parkinson’s disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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16 pages, 3194 KiB

Open AccessArticle

The Difference in Structural States between Canonical Proteins and Their Isoforms Established by Proteome-Wide Bioinformatics Analysis

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Biomolecules 2022, 12(11), 1610; https://doi.org/10.3390/biom12111610 - 01 Nov 2022

Cited by 5 | Viewed by 2123

Abstract

Alternative splicing is an important means of generating the protein diversity necessary for cellular functions. Hence, there is a growing interest in assessing the structural and functional impact of alternative protein isoforms. Typically, experimental studies are used to determine the structures of the canonical proteins ignoring the other isoforms. Therefore, there is still a large gap between abundant sequence information and meager structural data on these isoforms. During the last decade, significant progress has been achieved in the development of bioinformatics tools for structural and functional annotations of proteins. Moreover, the appearance of the AlphaFold program opened up the possibility to model a large number of high-confidence structures of the isoforms. In this study, using state-of-the-art tools, we performed in silico analysis of 58 eukaryotic proteomes. The evaluated structural states included structured domains, intrinsically disordered regions, aggregation-prone regions, and tandem repeats. Among other things, we found that the isoforms have fewer signal peptides, transmembrane regions, or tandem repeat regions in comparison with their canonical counterparts. This could change protein function and/or cellular localization. The AlphaFold modeling demonstrated that frequently isoforms, having differences with the canonical sequences, still can fold in similar structures though with significant structural rearrangements which can lead to changes of their functions. Based on the modeling, we suggested classification of the structural differences between canonical proteins and isoforms. Altogether, we can conclude that a majority of isoforms, similarly to the canonical proteins are under selective pressure for the functional roles. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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19 pages, 2321 KiB

Open AccessArticle

A Novel Tandem-Tag Purification Strategy for Challenging Disordered Proteins

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Biomolecules 2022, 12(11), 1566; https://doi.org/10.3390/biom12111566 - 26 Oct 2022

Cited by 1 | Viewed by 2209

Abstract

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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20 pages, 52022 KiB

Open AccessArticle

Sequence Properties of an Intramolecular Interaction that Inhibits p53 DNA Binding

Emily Gregory

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Gary W. Daughdrill

Biomolecules 2022, 12(11), 1558; https://doi.org/10.3390/biom12111558 - 25 Oct 2022

Cited by 3 | Viewed by 1416

Abstract

An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and ND WT for binding a consensus DNA sequence is −3.0 kcal/mol at physiological ionic strength. ΔΔG increased to −1.03 kcal/mol when acidic residues in TAD2 were changed to alanine (ND DE) and to −1.13 kcal/mol when all the nonpolar residues, including W53/F54, were changed to alanine (ND NP). These results indicate there is some cooperation between acidic, nonpolar, and aromatic residues from TAD2 to inhibit DNA binding. The dependence of DNA binding affinity on ionic strength was used to predict excess counterion release for binding both consensus and scrambled DNA sequences, which was smaller for ND WT and ND NP with consensus DNA and smaller for scrambled DNA overall. Using size exclusion chromatography, we show that the ND mutants have similar Stokes radii to ND WT suggesting the mutants disrupt autoinhibition without changing the global structure. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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12 pages, 1268 KiB

Open AccessArticle

Quantifying Coexistence Concentrations in Multi-Component Phase-Separating Systems Using Analytical HPLC

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Biomolecules 2022, 12(10), 1480; https://doi.org/10.3390/biom12101480 - 14 Oct 2022

Cited by 7 | Viewed by 1645

Abstract

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here, we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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20 pages, 6042 KiB

Open AccessEditor’s ChoiceArticle

Digging into the 3D Structure Predictions of AlphaFold2 with Low Confidence: Disorder and Beyond

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Biomolecules 2022, 12(10), 1467; https://doi.org/10.3390/biom12101467 - 13 Oct 2022

Cited by 8 | Viewed by 1843

Abstract

AlphaFold2 (AF2) has created a breakthrough in biology by providing three-dimensional structure models for whole-proteome sequences, with unprecedented levels of accuracy. In addition, the AF2 pLDDT score, related to the model confidence, has been shown to provide a good measure of residue-wise disorder. Here, we combined AF2 predictions with pyHCA, a tool we previously developed to identify foldable segments and estimate their order/disorder ratio, from a single protein sequence. We focused our analysis on the AF2 predictions available for 21 reference proteomes (AFDB v1), in particular on their long foldable segments (>30 amino acids) that exhibit characteristics of soluble domains, as estimated by pyHCA. Among these segments, we provided a global analysis of those with very low pLDDT values along their entire length and compared their characteristics to those of segments with very high pLDDT values. We highlighted cases containing conditional order, as well as cases that could form well-folded structures but escape the AF2 prediction due to a shallow multiple sequence alignment and/or undocumented structure or fold. AF2 and pyHCA can therefore be advantageously combined to unravel cryptic structural features in whole proteomes and to refine predictions for different flavors of disorder. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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19 pages, 3346 KiB

Open AccessArticle

Deciphering the Alphabet of Disorder—Glu and Asp Act Differently on Local but Not Global Properties

Mette Ahrensback Roesgaard

Rasmus Hartmann-Petersen

Kresten Lindorff-Larsen

and

Birthe B. Kragelund

Biomolecules 2022, 12(10), 1426; https://doi.org/10.3390/biom12101426 - 04 Oct 2022

Cited by 4 | Viewed by 1676

Abstract

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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26 pages, 4307 KiB

Open AccessArticle

The Ni(II)-Binding Activity of the Intrinsically Disordered Region of Human NDRG1, a Protein Involved in Cancer Development

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Biomolecules 2022, 12(9), 1272; https://doi.org/10.3390/biom12091272 - 09 Sep 2022

Cited by 2 | Viewed by 2130

Abstract

Nickel exposure is associated with tumors of the respiratory tract such as lung and nasal cancers, acting through still-uncharacterized mechanisms. Understanding the molecular basis of nickel-induced carcinogenesis requires unraveling the mode and the effects of Ni(II) binding to its intracellular targets. A possible Ni(II)-binding protein and a potential focus for cancer treatment is hNDRG1, a protein induced by Ni(II) through the hypoxia response pathway, whose expression correlates with higher cancer aggressiveness and resistance to chemotherapy in lung tissue. The protein sequence contains a unique C-terminal sequence of 83 residues (hNDRG1*C), featuring a three-times-repeated decapeptide, involved in metal binding, lipid interaction and post-translational phosphorylation. In the present work, the biochemical and biophysical characterization of unmodified hNDRG1*C was performed. Bioinformatic analysis assigned it to the family of the intrinsically disordered regions and the absence of secondary and tertiary structure was experimentally proven by circular dichroism and NMR. Isothermal titration calorimetry revealed the occurrence of a Ni(II)-binding event with micromolar affinity. Detailed information on the Ni(II)-binding site and on the residues involved was obtained in an extensive NMR study, revealing an octahedral paramagnetic metal coordination that does not cause any major change of the protein backbone, which is coherent with CD analysis. hNDRG1*C was found in a monomeric form by light-scattering experiments, while the full-length hNDRG1 monomer was found in equilibrium between the dimer and tetramer, both in solution and in human cell lines. The results are the first essential step for understanding the cellular function of hNDRG1*C at the molecular level, with potential future applications to clarify its role and the role of Ni(II) in cancer development. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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28 pages, 4791 KiB

Open AccessEditor’s ChoiceArticle

An Interpretable Machine-Learning Algorithm to Predict Disordered Protein Phase Separation Based on Biophysical Interactions

Hao Cai

Robert M. Vernon

and

Julie D. Forman-Kay

Biomolecules 2022, 12(8), 1131; https://doi.org/10.3390/biom12081131 - 17 Aug 2022

Cited by 14 | Viewed by 2127

Abstract

Protein phase separation is increasingly understood to be an important mechanism of biological organization and biomaterial formation. Intrinsically disordered protein regions (IDRs) are often significant drivers of protein phase separation. A number of protein phase-separation-prediction algorithms are available, with many being specific for particular classes of proteins and others providing results that are not amenable to the interpretation of the contributing biophysical interactions. Here, we describe LLPhyScore, a new predictor of IDR-driven phase separation, based on a broad set of physical interactions or features. LLPhyScore uses sequence-based statistics from the RCSB PDB database of folded structures for these interactions, and is trained on a manually curated set of phase-separation-driving proteins with different negative training sets including the PDB and human proteome. Competitive training for a variety of physical chemical interactions shows the greatest contribution of solvent contacts, disorder, hydrogen bonds, pi–pi contacts, and kinked beta-structures to the score, with electrostatics, cation–pi contacts, and the absence of a helical secondary structure also contributing. LLPhyScore has strong phase-separation-prediction recall statistics and enables a breakdown of the contribution from each physical feature to a sequence’s phase-separation propensity, while recognizing the interdependence of many of these features. The tool should be a valuable resource for guiding experiments and providing hypotheses for protein function in normal and pathological states, as well as for understanding how specificity emerges in defining individual biomolecular condensates. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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11 pages, 1692 KiB

Open AccessArticle

Folding and Binding Mechanisms of the SH2 Domain from Crkl

Valeria Pennacchietti

and

Biomolecules 2022, 12(8), 1014; https://doi.org/10.3390/biom12081014 - 22 Jul 2022

Cited by 4 | Viewed by 1499

Abstract

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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12 pages, 2617 KiB

Open AccessFeature PaperArticle

In-Silico Analysis of pH-Dependent Liquid-Liquid Phase Separation in Intrinsically Disordered Proteins

Carlos Pintado-Grima

Oriol Bárcenas

and

Salvador Ventura

Biomolecules 2022, 12(7), 974; https://doi.org/10.3390/biom12070974 - 12 Jul 2022

Cited by 7 | Viewed by 2052

Abstract

Intrinsically disordered proteins (IDPs) are essential players in the assembly of biomolecular condensates during liquid–liquid phase separation (LLPS). Disordered regions (IDRs) are significantly exposed to the solvent and, therefore, highly influenced by fluctuations in the microenvironment. Extrinsic factors, such as pH, modify the solubility and disorder state of IDPs, which in turn may impact the formation of liquid condensates. However, little attention has been paid to how the solution pH influences LLPS, despite knowing that this process is context-dependent. Here, we have conducted a large-scale in-silico analysis of pH-dependent solubility and disorder in IDRs known to be involved in LLPS (LLPS-DRs). We found that LLPS-DRs present maximum solubility around physiological pH, where LLPS often occurs, and identified significant differences in solubility and disorder between proteins that can phase-separate by themselves or those that require a partner. We also analyzed the effect of mutations in the resulting solubility profiles of LLPS-DRs and discussed how, as a general trend, LLPS-DRs display physicochemical properties that permit their LLPS at physiologically relevant pHs. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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13 pages, 1818 KiB

Open AccessEditor’s ChoiceArticle

NMR Reveals Specific Tracts within the Intrinsically Disordered Regions of the SARS-CoV-2 Nucleocapsid Protein Involved in RNA Encountering

and

Biomolecules 2022, 12(7), 929; https://doi.org/10.3390/biom12070929 - 02 Jul 2022

Cited by 8 | Viewed by 2106

Abstract

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. ¹³C-detected NMR experiments, acquired simultaneously to ¹H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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15 pages, 1507 KiB

Open AccessArticle

Compositional Bias of Intrinsically Disordered Proteins and Regions and Their Predictions

Bi Zhao

and

Lukasz Kurgan

Biomolecules 2022, 12(7), 888; https://doi.org/10.3390/biom12070888 - 25 Jun 2022

Cited by 10 | Viewed by 1537

Abstract

Intrinsically disordered regions (IDRs) carry out many cellular functions and vary in length and placement in protein sequences. This diversity leads to variations in the underlying compositional biases, which were demonstrated for the short vs. long IDRs. We analyze compositional biases across four classes of disorder: fully disordered proteins; short IDRs; long IDRs; and binding IDRs. We identify three distinct biases: for the fully disordered proteins, the short IDRs and the long and binding IDRs combined. We also investigate compositional bias for putative disorder produced by leading disorder predictors and find that it is similar to the bias of the native disorder. Interestingly, the accuracy of disorder predictions across different methods is correlated with the correctness of the compositional bias of their predictions highlighting the importance of the compositional bias. The predictive quality is relatively low for the disorder classes with compositional bias that is the most different from the “generic” disorder bias, while being much higher for the classes with the most similar bias. We discover that different predictors perform best across different classes of disorder. This suggests that no single predictor is universally best and motivates the development of new architectures that combine models that target specific disorder classes. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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15 pages, 6963 KiB

Open AccessArticle

Identification of Intrinsically Disordered Proteins and Regions in a Non-Model Insect Species Ostrinia nubilalis (Hbn.)

and

Biomolecules 2022, 12(4), 592; https://doi.org/10.3390/biom12040592 - 18 Apr 2022

Cited by 2 | Viewed by 1928

Abstract

Research in previous decades has shown that intrinsically disordered proteins (IDPs) and regions in proteins (IDRs) are as ubiquitous as highly ordered proteins. Despite this, research on IDPs and IDRs still has many gaps left to fill. Here, we present an approach that combines wet lab methods with bioinformatics tools to identify and analyze intrinsically disordered proteins in a non-model insect species that is cold-hardy. Due to their known resilience to the effects of extreme temperatures, these proteins likely play important roles in this insect’s adaptive mechanisms to sub-zero temperatures. The approach involves IDP enrichment by sample heating and double-digestion of proteins, followed by peptide and protein identification. Next, proteins are bioinformatically analyzed for disorder content, presence of long disordered regions, amino acid composition, and processes they are involved in. Finally, IDP detection is validated with an in-house 2D PAGE. In total, 608 unique proteins were identified, with 39 being mostly disordered, 100 partially disordered, 95 nearly ordered, and 374 ordered. One-third contain at least one long disordered segment. Functional information was available for only 90 proteins with intrinsic disorders out of 312 characterized proteins. Around half of the 90 proteins are cytoskeletal elements or involved in translational processes. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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15 pages, 2438 KiB

Open AccessEditor’s ChoiceArticle

Distribution of Charged Residues Affects the Average Size and Shape of Intrinsically Disordered Proteins

and

Biomolecules 2022, 12(4), 561; https://doi.org/10.3390/biom12040561 - 09 Apr 2022

Cited by 8 | Viewed by 2504

Abstract

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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Review

Jump to: Editorial, Research

17 pages, 2022 KiB

Open AccessReview

Topological Considerations in Biomolecular Condensation

Debapriya Das

and

Ashok A. Deniz

Biomolecules 2023, 13(1), 151; https://doi.org/10.3390/biom13010151 - 11 Jan 2023

Cited by 2 | Viewed by 2246

Abstract

Biomolecular condensation and phase separation are increasingly understood to play crucial roles in cellular compartmentalization and spatiotemporal regulation of cell machinery implicated in function and pathology. A key aspect of current research is to gain insight into the underlying physical mechanisms of these processes. Accordingly, concepts of soft matter and polymer physics, the thermodynamics of mixing, and material science have been utilized for understanding condensation mechanisms of multivalent macromolecules resulting in viscoelastic mesoscopic supramolecular assemblies. Here, we focus on two topological concepts that have recently been providing key mechanistic understanding in the field. First, we will discuss how percolation provides a network-topology-related framework that offers an interesting paradigm to understand the complex networking of dense ‘connected’ condensate structures and, therefore, their phase behavior. Second, we will discuss the idea of entanglement as another topological concept that has deep roots in polymer physics and important implications for biomolecular condensates. We will first review some historical developments and fundamentals of these concepts, then we will discuss current advancements and recent examples. Our discussion ends with a few open questions and the challenges to address them, hinting at unveiling fresh possibilities for the modification of existing knowledge as well as the development of new concepts relevant to condensate science. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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19 pages, 1613 KiB

Open AccessFeature PaperReview

Illuminating Intrinsically Disordered Proteins with Integrative Structural Biology

and

Biomolecules 2023, 13(1), 124; https://doi.org/10.3390/biom13010124 - 07 Jan 2023

Cited by 3 | Viewed by 2084

Abstract

Intense study of intrinsically disordered proteins (IDPs) did not begin in earnest until the late 1990s when a few groups, working independently, convinced the community that these ‘weird’ proteins could have important functions. Over the past two decades, it has become clear that IDPs play critical roles in a multitude of biological phenomena with prominent examples including coordination in signaling hubs, enabling gene regulation, and regulating ion channels, just to name a few. One contributing factor that delayed appreciation of IDP functional significance is the experimental difficulty in characterizing their dynamic conformations. The combined application of multiple methods, termed integrative structural biology, has emerged as an essential approach to understanding IDP phenomena. Here, we review some of the recent applications of the integrative structural biology philosophy to study IDPs. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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36 pages, 2462 KiB

Open AccessReview

Reorganization of Cell Compartmentalization Induced by Stress

Anna S. Fefilova

Iuliia A. Antifeeva

Anastasia A. Gavrilova

Konstantin K. Turoverov

Irina M. Kuznetsova

and

Alexander V. Fonin

Biomolecules 2022, 12(10), 1441; https://doi.org/10.3390/biom12101441 - 08 Oct 2022

Cited by 2 | Viewed by 2834

Abstract

The discovery of intrinsically disordered proteins (IDPs) that do not have an ordered structure and nevertheless perform essential functions has opened a new era in the understanding of cellular compartmentalization. It threw the bridge from the mostly mechanistic model of the organization of the living matter to the idea of highly dynamic and functional “soft matter”. This paradigm is based on the notion of the major role of liquid-liquid phase separation (LLPS) of biopolymers in the spatial-temporal organization of intracellular space. The LLPS leads to the formation of self-assembled membrane-less organelles (MLOs). MLOs are multicomponent and multifunctional biological condensates, highly dynamic in structure and composition, that allow them to fine-tune the regulation of various intracellular processes. IDPs play a central role in the assembly and functioning of MLOs. The LLPS importance for the regulation of chemical reactions inside the cell is clearly illustrated by the reorganization of the intracellular space during stress response. As a reaction to various types of stresses, stress-induced MLOs appear in the cell, enabling the preservation of the genetic and protein material during unfavourable conditions. In addition, stress causes structural, functional, and compositional changes in the MLOs permanently present inside the cells. In this review, we describe the assembly of stress-induced MLOs and the stress-induced modification of existing MLOs in eukaryotes, yeasts, and prokaryotes in response to various stress factors. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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20 pages, 2533 KiB

Open AccessReview

The Role of Intrinsically Disordered Proteins in Liquid–Liquid Phase Separation during Calcium Carbonate Biomineralization

and

Biomolecules 2022, 12(9), 1266; https://doi.org/10.3390/biom12091266 - 09 Sep 2022

Cited by 7 | Viewed by 2437

Abstract

Some animal organs contain mineralized tissues. These so-called hard tissues are mostly deposits of calcium salts, usually in the form of calcium phosphate or calcium carbonate. Examples of this include fish otoliths and mammalian otoconia, which are found in the inner ear, and they are an essential part of the sensory system that maintains body balance. The composition of ear stones is quite well known, but the role of individual components in the nucleation and growth of these biominerals is enigmatic. It is sure that intrinsically disordered proteins (IDPs) play an important role in this aspect. They have an impact on the shape and size of otoliths. It seems probable that IDPs, with their inherent ability to phase separate, also play a role in nucleation processes. This review discusses the major theories on the mechanisms of biomineral nucleation with a focus on the importance of protein-driven liquid–liquid phase separation (LLPS). It also presents the current understanding of the role of IDPs in the formation of calcium carbonate biominerals and predicts their potential ability to drive LLPS. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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16 pages, 2102 KiB

Open AccessReview

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12

Cassy Gérard

Frédéric Carrière

Véronique Receveur-Bréchot

Hélène Launay

and

Brigitte Gontero

Biomolecules 2022, 12(8), 1047; https://doi.org/10.3390/biom12081047 - 28 Jul 2022

Cited by 6 | Viewed by 1626

Abstract

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO₂ assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have. Full article

(This article belongs to the Special Issue Physics of Protein Folding, Misfolding, and Intrinsic Disorder: A Themed Issue in Honour of Professor Vladimir Uversky on the Occasion of His 60th Birthday)

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