Natively Unstructured Loops Differ from Other Loops

Natively unstructured or disordered protein regions may increase the functional complexity of an organism; they are particularly abundant in eukaryotes and often evade structure determination. Many computational methods predict unstructured regions by training on outliers in otherwise well-ordered structures. Here, we introduce an approach that uses a neural network in a very different and novel way. We hypothesize that very long contiguous segments with nonregular secondary structure (NORS regions) differ significantly from regular, well-structured loops, and that a method detecting such features could predict natively unstructured regions. Training our new method, NORSnet, on predicted information rather than on experimental data yielded three major advantages: it removed the overlap between testing and training, it systematically covered entire proteomes, and it explicitly focused on one particular aspect of unstructured regions with a simple structural interpretation, namely that they are loops. Our hypothesis was correct: well-structured and unstructured loops differ so substantially that NORSnet succeeded in their distinction. Benchmarks on previously used and new experimental data of unstructured regions revealed that NORSnet performed very well. Although it was not the best single prediction method, NORSnet was sufficiently accurate to flag unstructured regions in proteins that were previously not annotated. In one application, NORSnet revealed previously undetected unstructured regions in putative targets for structural genomics and may thereby contribute to increasing structural coverage of large eukaryotic families. NORSnet found unstructured regions more often in domain boundaries than expected at random. In another application, we estimated that 50%–70% of all worm proteins observed to have more than seven protein–protein interaction partners have unstructured regions. The comparative analysis between NORSnet and DISOPRED2 suggested that long unstructured loops are a major part of unstructured regions in molecular networks

Computational analysis of folding and mutation properties of C5 domain from Myosin binding protein C

Thermal folding Molecular Dynamics simulations of the domain C5 from Myosin Binding Protein C were performed using a native-centric model to study the role of three mutations related to Familial Hypertrophic Cardiomyopathy. Mutation of Asn755 causes the largest shift of the folding temperature, and the residue is located in the CFGA' beta-sheet featuring the highest Phi-values. The mutation thus appears to reduce the thermodynamic stability in agreement with experimental data. The mutations on Arg654 and Arg668, conversely, cause a little change in the folding temperature and they reside in the low Phi-value BDE beta-sheet, so that their pathologic role cannot be related to impairment of the folding process but possibly to the binding with target molecules. As the typical signature of Domain C5 is the presence of a longer and destabilizing CD-loop with respect to the other Ig-like domains we completed the work with a bioinformatic analysis of this loop showing a high density of negative charge and low hydrophobicity. This indicates the CD-loop as a natively unfolded sequence with a likely coupling between folding and ligand binding.Comment: RevTeX, 10 pages, 9 eps-figure

Dissecting the oxidative folding of circular cystine knot miniproteins

Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Möbius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the folding process. Overall, these mechanistic findings on the folding of cyclotides are potentially valuable for the design of new drug leads. Copyright Mary Ann Liebert, Inc

Coarse-grained models for Protein Folding and Function

The plan of this thesis is as follows: in the next chapter (chapter 1) we review the main properties of globular proteins, in particular focusing on the state of the art of protein folding and design. Then we describe in detail the simple model for folding adopted throughout the present work (chapter 2). Chapter 3 shows the further modeling introduced to handle the specific subject of disordered proteins, with full explanation of all parameters used and with some possible interpretation of the results obtained. In the last chapter of this work (chapter 4) we present a study on the near equilibrium dynamics of two small proteins in the family of truncated hemoglobins, developed under the framework of a Gaussian network approach

Disease-Associated Mutations Disrupt Functionally Important Regions of Intrinsic Protein Disorder

The effects of disease mutations on protein structure and function have been extensively investigated, and many predictors of the functional impact of single amino acid substitutions are publicly available. The majority of these predictors are based on protein structure and evolutionary conservation, following the assumption that disease mutations predominantly affect folded and conserved protein regions. However, the prevalence of the intrinsically disordered proteins (IDPs) and regions (IDRs) in the human proteome together with their lack of fixed structure and low sequence conservation raise a question about the impact of disease mutations in IDRs. Here, we investigate annotated missense disease mutations and show that 21.7% of them are located within such intrinsically disordered regions. We further demonstrate that 20% of disease mutations in IDRs cause local disorder-to-order transitions, which represents a 1.7–2.7 fold increase compared to annotated polymorphisms and neutral evolutionary substitutions, respectively. Secondary structure predictions show elevated rates of transition from helices and strands into loops and vice versa in the disease mutations dataset. Disease disorder-to-order mutations also influence predicted molecular recognition features (MoRFs) more often than the control mutations. The repertoire of disorder-to-order transition mutations is limited, with five most frequent mutations (R→W, R→C, E→K, R→H, R→Q) collectively accounting for 44% of all deleterious disorder-to-order transitions. As a proof of concept, we performed accelerated molecular dynamics simulations on a deleterious disorder-to-order transition mutation of tumor protein p63 and, in agreement with our predictions, observed an increased α-helical propensity of the region harboring the mutation. Our findings highlight the importance of mutations in IDRs and refine the traditional structure-centric view of disease mutations. The results of this study offer a new perspective on the role of mutations in disease, with implications for improving predictors of the functional impact of missense mutations

Protein secondary structure appears to be robust under in silico evolution while protein disorder appears not to be

Motivation: The mutation of amino acids often impacts protein function and structure. Mutations without negative effect sustain evolutionary pressure. We study a particular aspect of structural robustness with respect to mutations: regular protein secondary structure and natively unstructured (intrinsically disordered) regions. Is the formation of regular secondary structure an intrinsic feature of amino acid sequences, or is it a feature that is lost upon mutation and is maintained by evolution against the odds? Similarly, is disorder an intrinsic sequence feature or is it difficult to maintain? To tackle these questions, we in silico mutated native protein sequences into random sequence-like ensembles and monitored the change in predicted secondary structure and disorder

The Role of Disordered Ribosomal Protein Extensions in the Early Steps of Eubacterial 50 S Ribosomal Subunit Assembly

Although during the past decade research has shown the functional importance of disorder in proteins, many of the structural and dynamics properties of intrinsically unstructured proteins (IUPs) remain to be elucidated. This review is focused on the role of the extensions of the ribosomal proteins in the early steps of the assembly of the eubacterial 50 S subunit. The recent crystallographic structures of the ribosomal particles have revealed the picture of a complex assembly pathway that condenses the rRNA and the ribosomal proteins into active ribosomes. However, little is know about the molecular mechanisms of this process. It is thought that the long basic r-protein extensions that penetrate deeply into the subunit cores play a key role through disorder-order transitions and/or co-folding mechanisms. A current view is that such structural transitions may facilitate the proper rRNA folding. In this paper, the structures of the proteins L3, L4, L13, L20, L22 and L24 that have been experimentally found to be essential for the first steps of ribosome assembly have been compared. On the basis of their structural and dynamics properties, three categories of extensions have been identified. Each of them seems to play a distinct function. Among them, only the coil-helix transition that occurs in a phylogenetically conserved cluster of basic residues of the L20 extension appears to be strictly required for the large subunit assembly in eubacteria. The role of α helix-coil transitions in 23 S RNA folding is discussed in the light of the calcium binding protein calmodulin that shares many structural and dynamics properties with L20

Performance of Protein Disorder Prediction Programs on Amino Acid Substitutions

Many proteins contain intrinsically disordered regions, which may be crucial for function, but on the other hand be related to the pathogenicity of variants. Prediction programs have been developed to detect disordered regions from sequences and used to predict the consequences of variants, although their performance for this task has not been assessed. We tested the performance of protein disorder prediction programs in detecting changes to disorder caused by amino acid substitutions. We assessed the performance of 29 protein disorder predictors and versions with 101 amino acid substitutions, whose effects have been experimentally validated. Disorder predictors detected the true positives at most with 6% success rate and true negatives with 34% rate for variants. The corresponding rates for the wild-type forms are 7% and 90%, respectively. The analysis revealed that disorder programs cannot reliably predict the effects of substitutions; consequently, the tested methods, and possibly similar programs, cannot be recommended for variant analysis without other information indicating to the relevance of disorder. These results inspired us to develop a new method, PON-Diso (http://structure.bmc.lu.se/PON-Diso), for disorder-related amino acid substitutions. With 50% success rate for independent test set and 70.5% rate in cross-validation, it outperforms the evaluated methods