Papers by Vladimir Uversky
Protein Engineering Design and Selection, 2005
It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone... more It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca(2+)-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca(2+)-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.
FEBS Journal, 2005
Proteins participate in complex sets of interactions that represent the mechanistic foundation fo... more Proteins participate in complex sets of interactions that represent the mechanistic foundation for much of the physiology and function of the cell. These protein-protein interactions are organized into exquisitely complex networks. The architecture of protein-protein interaction networks was recently proposed to be scale-free, with most of the proteins having only one or two connections but with relatively fewer 'hubs' possessing tens, hundreds or more links. The high level of hub connectivity must somehow be reflected in protein structure. What structural quality of hub proteins enables them to interact with large numbers of diverse targets? One possibility would be to employ binding regions that have the ability to bind multiple, structurally diverse partners. This trait can be imparted by the incorporation of intrinsic disorder in one or both partners. To illustrate the value of such contributions, this review examines the roles of intrinsic disorder in protein network architecture. We show that there are three general ways that intrinsic disorder can contribute: First, intrinsic disorder can serve as the structural basis for hub protein promiscuity; secondly, intrinsically disordered proteins can bind to structured hub proteins; and thirdly, intrinsic disorder can provide flexible linkers between functional domains with the linkers enabling mechanisms that facilitate binding diversity. An important research direction will be to determine what fraction of protein-protein interaction in regulatory networks relies on intrinsic disorder.
Biochemistry, 2005
Intrinsically disordered proteins and regions carry out varied and vital cellular functions. Prot... more Intrinsically disordered proteins and regions carry out varied and vital cellular functions. Proteins with disordered regions are especially common in eukaryotic cells, with a subset of these proteins being mostly disordered, e.g., with more disordered than ordered residues. Two distinct methods have been previously described for using amino acid sequences to predict which proteins are likely to be mostly disordered. These methods are based on the net charge-hydropathy distribution and disorder prediction score distribution. Each of these methods is reexamined, and the prediction results are compared herein. A new prediction method based on consensus is described. Application of the consensus method to whole genomes reveals that approximately 4.5% of Yersinia pestis, 5% of Escherichia coli K12, 6% of Archaeoglobus fulgidus, 8% of Methanobacterium thermoautotrophicum, 23% of Arabidopsis thaliana, and 28% of Mus musculus proteins are mostly disordered. The unexpectedly high frequency of mostly disordered proteins in eukaryotes has important implications both for large-scale, high-throughput projects and also for focused experiments aimed at determination of protein structure and function.
Journal of Biomolecular Structure and Dynamics, 2007
The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB conta... more The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only approximately 7% of proteins are observed in the corresponding PDB structures, and only approximately 25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, "Observed" (which correspond to structured regions), "Not observed" (regions with missing electron density, potentially disordered), "Uncharacterized," and "Ambiguous," depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a 'fragment' or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. "Non-observed," "Ambiguous," and "Uncharacterized" regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR(R) VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the…
Biochemistry, 2005
Many protein-protein and protein-nucleic acid interactions involve coupled folding and binding of... more Many protein-protein and protein-nucleic acid interactions involve coupled folding and binding of at least one of the partners. Here, we propose a protein structural element or feature that mediates the binding events of initially disordered regions. This element ...
Journal of Proteome Research, 2007
Molecular Recognition Features (MoRFs) are short, interaction-prone segments of protein disorder ... more Molecular Recognition Features (MoRFs) are short, interaction-prone segments of protein disorder that undergo disorder-to-order transitions upon specific binding, representing a specific class of intrinsically disordered regions that exhibit molecular recognition and binding functions. MoRFs are common in various proteomes and occupy a unique structural and functional niche in which function is a direct consequence of intrinsic disorder. Example MoRFs collected from the Protein Data Bank (PDB) have been divided into three subtypes according to their structures in the bound state: α-MoRFs form α-helices, β-MoRFs form β-strands, and ι-MoRFs form structures without a regular pattern of backbone hydrogen bonds. These example MoRFs were indicated to be intrinsically disordered in the absence of their binding partners by several criteria. In this study we used several geometric and physiochemical criteria to examine the properties of 62 α-, 20 βand 176 ι-MoRF complex structures. Interface residues were examined by calculating differences in accessible surface area between the complex and isolated monomers. The compositions and physiochemical properties of MoRF and MoRF partner interface residues were compared to the interface residues of homodimers, heterodimers, and antigen-antibody complexes. Our analysis indicates that there are significant differences in residue composition and several geometric and physicochemical properties that can be used to discriminate, with a high degree of accuracy, between various interfaces in protein interaction datasets. Implications of these findings for the development of MoRF-partner interaction predictors are discussed. In addition, structural changes upon MoRF-to-partner complex formation were examined for several illustrative examples.
Journal of Proteome Research, 2005
A method to enrich cell extracts in totally unfolded proteins was investigated. A literature sear... more A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment where also shown experimentally to be totally disordered. A near 100 000-fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble E. coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver-stained 2-D SDS-PAGE gels loaded with 10 microg of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than 3-fold higher yield. A substantial yield of unprecipitated protein was obtained after 3% TCA treatment, suggesting that the common use of TCA precipitation prior to 2-D gel analysis may result in loss of unstructured protein due to their failure to precipitate. Our preliminary analysis suggests that treating total protein extracts with 3-5% PCA and determining the identities of soluble proteins could be the starting point for uncovering unfoldomes (the complement of unstructured proteins in a given proteome). The 100 000-fold reduction in yield and concomitant reduction in number of proteins achieved by 5% PCA treatment produced a fraction suitable for analysis in its entirety using standard proteomic techniques. In this way, large numbers of totally unstructured proteins could be identified with minimal effort.
Nucleic Acids Research, 2007
The Database of Protein Disorder (DisProt) links structure and function information for intrinsic... more The Database of Protein Disorder (DisProt) links structure and function information for intrinsically disordered proteins (IDPs). Intrinsically disordered proteins do not form a fixed three-dimensional structure under physiological conditions, either in their entireties or in segments or regions. We define IDP as a protein that contains at least one experimentally determined disordered region. Although lacking fixed structure, IDPs and regions carry out important biological functions, being typically involved in regulation, signaling and control. Such functions can involve high-specificity low-affinity interactions, the multiple binding of one protein to many partners and the multiple binding of many proteins to one partner. These three features are all enabled and enhanced by protein intrinsic disorder. One of the major hindrances in the study of IDPs has been the lack of organized information. DisProt was developed to enable IDP research by collecting and organizing knowledge regarding the experimental characterization and the functional associations of IDPs. In addition to being a unique source of biological information, DisProt opens doors for a plethora of bioinformatics studies. DisProt is openly available at
Trends in Biotechnology, 2006
Despite substantial increases in research funding by the pharmaceutical industry, drug discovery ... more Despite substantial increases in research funding by the pharmaceutical industry, drug discovery rates seem to have reached a plateau or perhaps are even declining, suggesting the need for new strategies. Protein-protein interactions have long been thought to provide interesting drug discovery targets, but the development of small molecules that modulate such interactions has so far achieved a low success rate. In contrast to this historic trend, a few recent successes raise hopes for routinely identifying druggable protein-protein interactions. In this Opinion article, we point out the importance of coupled binding and folding for protein-protein signalling interactions generally, and from this and associated observations, we develop a new strategy for identifying protein-protein interactions that would be particularly promising targets for modulation by small molecules. This novel strategy, based on intrinsically disordered protein, has the potential to increase significantly the discovery rate for new molecule entities.
Protein Folding Handbook, 2005
... lacks specific tertiary structures, having instead regions of non-rigid side chain packing th... more ... lacks specific tertiary structures, having instead regions of non-rigid side chain packing that leads ... In one of these studies, reduced spectral density mapping was used to help ... and the intrinsically disordered N-terminal DNA-binding domain were identical within experimental error ...
Proceedings of The National Academy of Sciences, 2006
Alternative splicing of pre-mRNA generates two or more protein isoforms from a single gene, there... more Alternative splicing of pre-mRNA generates two or more protein isoforms from a single gene, thereby contributing to protein diversity. Despite intensive efforts, an understanding of the protein structure-function implications of alternative splicing is still lacking. Intrinsic disorder, which is a lack of equilibrium 3D structure under physiological conditions, may provide this understanding. Intrinsic disorder is a common phenomenon, particularly in
Biochemistry, 2002
... John Goers, § Sergei E. Permyakov, Eugene A. Permyakov, Vladimir N. Uversky,* and Anthony... more ... John Goers, § Sergei E. Permyakov, Eugene A. Permyakov, Vladimir N. Uversky,* and Anthony L. Fink . Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, Institute for Biological ...
Protein …, Jan 1, 2010
Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, par... more Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N-terminal and large natively unfolded Cterminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two-hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N-terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C-terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or ''hubs'' are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein-protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.
Molecular BioSystems, 2011
Although the members of the largest subfamily of the EF-hand proteins, S100 proteins, are evoluti... more Although the members of the largest subfamily of the EF-hand proteins, S100 proteins, are evolutionarily young, their functional diversity is extremely broad, partly due to their ability to adapt to various targets. This feature is a hallmark of intrinsically disordered proteins (IDPs), but none of the S100 proteins are recognized as IDPs. S100 are predicted to be enriched in intrinsic disorder, with 62% of them being predicted to be disordered by at least one of the predictors: 31% are recognized as 'molten globules' and 15% are shown to be in extended disordered form. The disorder level of predicted disordered S100 regions is conserved compared to that of more structured regions. The central disordered stretch corresponds to the major part of pseudo EF-hand loop, helix II, hinge region, and an initial part of helix III. It contains about half of known sites of enzymatic post-translational modifications (PTMs), confirming that this region can be flexible in vivo. Most of the internal residues missing in tertiary structures belong to the hinge. Both hinge and pseudo EF-hand loop correspond to the local maxima of the PONDR® VSL2 score and are shown to be evolutionary hotspots, leading to gain of new functional properties. The action of PTMs is shown to be destabilizing, in contrast with the effect of metal-binding or S100 dimerization. Formation of the S100 heterodimers relies on the interplay between the structural rigidity of one of the S100 monomers and the flexibility of another monomer. The ordered regions dominate in the S100 homodimerization sites. Target-binding sites generally consist of distant regions, drastically differing in their disorder level. The disordered region comprising most of the hinge and the N-terminal half of helix III is virtually not involved into dimerization, being intended solely for target recognition. The structural flexibility of this region is essential for recognition of diverse target proteins. At least 86% of multiple interactions of S100 proteins with binding partners are attributed to the S100 proteins predicted to be disordered. Overall, the intrinsic disorder is inherent to many S100 proteins and is vital for activity and functional diversity of the family.
Biochemical and Biophysical Research Communications, 2000
i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SA... more i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein.
Protein Engineering Design and Selection, 2000
dependent inhibition of rhodopsin kinase Sergey V.Shulga-Morskoy 3 , Andrey M.Alekseev 3 , and th... more dependent inhibition of rhodopsin kinase Sergey V.Shulga-Morskoy 3 , Andrey M.Alekseev 3 , and thus participates in the regulation of the photoresponse Dmitry V.Zinchenko 3 , Valery M.Lipkin 3 , duration. The amino terminus of recoverin is heterogeneously Pavel P.Philippov 2 , Vladimir N.Uversky 1,4,5 and fatty acid acylated (mainly myristoylated) (Dizhoor et al., Eugene A.Permyakov 1 1992). This modification enhances the recoverin inhibitory efficiency with respect to rhodopsin kinase .
Molecular Brain Research, 2005
Substantial evidence suggests that the aggregation of the presynaptic protein a-synuclein is a ke... more Substantial evidence suggests that the aggregation of the presynaptic protein a-synuclein is a key step in the etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying a-synuclein aggregation remain unknown, oxidative stress has been implicated in the pathogenesis of PD. Here, we report the effects of tyrosine nitration on the propensity of human recombinant a-synuclein to fibrillate in vitro. The properties of nitrated a-synuclein were investigated using a variety of biophysical and biochemical techniques, which revealed that nitration led to formation of a partially folded conformation with increased secondary structure relative to the intrinsically disordered structure of the monomer, and to oligomerization at neutral pH. The degree of self-association was concentration-dependent, but at 1 mg/mL, nitrated a-synuclein was predominantly an octamer. At low pH, small-angle X-ray scattering data indicated that the nitrated protein was monomeric. a-Synuclein fibrillation at neutral pH was completely inhibited by nitrotyrosination and is attributed to the formation of stable soluble oligomers. The presence of heparin or metals did not overcome the inhibition; however, the inhibitory effect was eliminated at low pH. The addition of nitrated a-synuclein inhibited fibrillation of non-modified a-synuclein at neutral pH. Potential implications of these findings to the etiology of Parkinson's disease are discussed. D
Journal of Molecular Biology, 2005
Protein misfolding is conformational transition dramatically facilitating the assembly of protein... more Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, a-synuclein, amyloid b-peptide (Ab) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for a-synuclein and Ab revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly b-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.
Journal of Biological Chemistry, 2010
The plant growth-repressing DELLA proteins (DELLAs) are known to represent a convergence point in... more The plant growth-repressing DELLA proteins (DELLAs) are known to represent a convergence point in integration of multiple developmental and environmental signals in planta, one of which is hormone gibberellic acid (GA). Binding of the liganded GA receptor (GID1/GA) to the N-terminal domain of DELLAs is required for GA-induced degradation of DELLAs via the ubiquitin-proteasome pathway, thus derepressing plant growth. However, the conformational changes of DELLAs upon binding to GID1/GA, which are the key to understanding the precise mechanism of GID1/GA-mediated degradation of DELLAs, remain unclear. Using biophysical, biochemical, and bioinformatics approaches, we demonstrated for the first time that the unbound N-terminal domains of DELLAs are intrinsically unstructured proteins under physiological conditions. Within the intrinsically disordered N-terminal domain of DELLAs, we have identified several molecular recognition features, sequences known to undergo disorder-to-order transitions upon binding to interacting proteins in intrinsically unstructured proteins. In accordance with the molecular recognition feature analyses, we have observed the binding-induced folding of N-terminal domains of DELLAs upon interaction with AtGID1/GA. Our results also indicate that DELLA proteins can be divided into two subgroups in terms of their molecular compactness and their interactions with monoclonal antibodies.
Metallomics, 2011
Neurodegenerative diseases constitute a set of pathological conditions originating from the slow,... more Neurodegenerative diseases constitute a set of pathological conditions originating from the slow, irreversible, and systematic cell loss within the various regions of the brain and/or the spinal cord. Depending on the affected region, the outcomes of the neurodegeneration are very broad and diverse, ranging from the problems with movements to dementia. Some neurodegenerative diseases are associated with protein misfolding and aggregation. Many proteins that misfold in human neurodegenerative diseases are intrinsically disordered; i.e., they lack a stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) functionally complement ordered proteins, being typically involved in regulation and signaling. There is accumulating evidence that altered metal homeostasis may be related to the progression of neurodegenerative diseases. This review examines the effects of metal ion binding on the aggregation pathways of IDPs found in neurodegenerative diseases.
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Papers by Vladimir Uversky
very different structures in the bound state, or where multiple unrelated IDPs/IDPRs bind to one partner. Binding functions of IDPs and IDPRs are controlled by various means, such as numerous posttranslational modifications and alternative splicing. Some of the aspects of the intrinsic disorder-based protein interactions and modes of their regulation are considered in this review.