Approaches to predicting effects of single amino acid substitutions on the function of a protein

The relative activities of 313 mutants of the gene V protein of bacteriophage f1, assayed in vivo, have been used to evaluate two approaches to predicting the effects of single amino acid substitutions on the function of a protein. First, we tested methods that only depend on the properties of the wild-type and substituting amino acids. None of the properties or measures of the functional equivalence of amino acids we tested, including the frequency of exchange of amino acids among homologous proteins as well as changes in side-chain size, hydrophobicity, and charge, were found to be more than weakly correlated with the activities of mutants. The principal reason for this poor correlation was found to be that the effect of a particular substitution varies considerably from site to site. We then tested an approach using the activities of several mutants with substitutions at a site to predict the activity of another mutant, and we find that this is a relatively good indicator of whether the other mutant at that site will be functional. A predictive scheme was developed that combines the weak information from the models depending on the properties of the wild-type and substituting amino acids with the stronger information from the tolerance of a site to substitution. Although this scheme requires no knowledge of the structure of a mutant protein, it is useful in predicting the activities of mutants.     

Effect of single amino acid substitutions at the same position on stability of a two-domain protein

In order to elucidate the role of individual amino acid residues on the conformational stability of a protein, the stabilities of the wild-type tryptophan synthase α-subunit from Escherichia coil and its five mutant proteins substituted by single amino acid residues at the same position 49 were compared. The five mutant proteins have glutamine, methionine, valine, serine, or tyrosine in place of glutamic acid of the wild-type protein at position 49. Denaturation of these proteins, which consist of two domains, by guanidine hydrochloride can be analyzed as a two-step process. We obtained the equilibrium constants between the native and the denatured forms and between the native and the stable intermediate forms for the above six proteins in the absence of denaturant at three pH values.     

Effect of amino acid substitutions on conformational stability of a protein

This paper reviews studies on thermostable proteins from thermophilic bacteria and on mutant proteins of human hemoglobin, tryptophan synthase α-subunit of E. coli, T₄ phage lysozyme, and phage λ repressor with respect to the role of the consisting amino acid residues in stabilization of conformation. The stability of a protein is easily affected by single amino acid substitutions, by which the protein undergoes change(s) of one or more of the following: a hydrogen bond, a salt bridge, a hydrophobic interaction, the volume of the residue, a disulfide bond, or the relative position of two aromatic rings.     

Machine learning methods for predicting protein structure from single sequences

Recent breakthroughs in protein structure prediction have increasingly relied on the use of deep neural networks. These recent methods are notable in that they produce 3-D atomic coordinates as a direct output of the networks, a feature which presents many advantages. Although most techniques of this type make use of multiple sequence alignments as their primary input, a new wave of methods have attempted to use just single sequences as the input. We discuss the make-up and operating principles of these models, and highlight new developments in these areas, as well as areas for future development.     

Sequence feature-based prediction of protein stability changes upon amino acid substitutions

Background

Protein destabilization is a common mechanism by which amino acid substitutions cause human diseases. Although several machine learning methods have been reported for predicting protein stability changes upon amino acid substitutions, the previous studies did not utilize relevant sequence features representing biological knowledge for classifier construction.

Results

In this study, a new machine learning method has been developed for sequence feature-based prediction of protein stability changes upon amino acid substitutions. Support vector machines were trained with data from experimental studies on the free energy change of protein stability upon mutations. To construct accurate classifiers, twenty sequence features were examined for input vector encoding. It was shown that classifier performance varied significantly by using different sequence features. The most accurate classifier in this study was constructed using a combination of six sequence features. This classifier achieved an overall accuracy of 84.59% with 70.29% sensitivity and 90.98% specificity.

Conclusions

Relevant sequence features can be used to accurately predict protein stability changes upon amino acid substitutions. Predictive results at this level of accuracy may provide useful information to distinguish between deleterious and tolerant alterations in disease candidate genes. To make the classifier accessible to the genetics research community, we have developed a new web server, called MuStab (http://bioinfo.ggc.org/mustab/).

     

Effect of conserved intersubunit amino acid substitutions on Hfq protein structure and stability

Hfq is a thermostable RNA-binding bacterial protein that forms a uniquely shaped homohexamer. Based on sequence and structural similarity, Hfq belongs to the like-Sm (LSm) protein family. In spite of a rather high degree of homology between archaeal and eukaryotic LSm proteins, their quaternary structure is different, usually consisting of five to eight monomers. In this work, the importance of conserved intersubunit hydrogen bonds for the Hfq spatial organization was tested. The structures and stabilities for the Gln8Ala, Asn28Ala, Asp40Ala, and Tyr55Ala Hfq mutants were determined. All these proteins have the same hexamer organization, but their stability is different. Elimination of a single intersubunit hydrogen bond due to Gln8Ala, Asp40Ala, and Tyr55Ala substitutions results in decreased stability of the Hfq hexamer. Tyr55Ala Hfq as well as the earlier studied His57Ala Hfq has reduced protein thermostability, which seems to correspond to an opening of the protein hydrophobic core.     

Prediction of protein disorder on amino acid substitutions

Intrinsically disordered regions of proteins are known to have many functional roles in cell signaling and regulatory pathways. The altered expression of these proteins due to mutations is associated with various diseases. Currently, most of the available methods focus on predicting the disordered proteins or the disordered regions in a protein. On the other hand, methods developed for predicting protein disorder on mutation showed a poor performance with a maximum accuracy of 70%. Hence, in this work, we have developed a novel method to classify the disorder-related amino acid substitutions using amino acid properties, substitution matrices, and the effect of neighboring residues that showed an accuracy of 90.0% with a sensitivity and specificity of 94.9 and 80.6%, respectively, in 10-fold cross-validation. The method was evaluated with a test set of 20% data using 10 iterations, which showed an average accuracy of 88.9%. Furthermore, we systematically analyzed the features responsible for the better performance of our method and observed that neighboring residues play an important role in defining the disorder of a given residue in a protein sequence. We have developed a prediction server to identify disorder-related mutations, and it is available at http://www.iitm.ac.in/bioinfo/DIM_Pred/.     

Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

The functional (synthetic) activity of blood lymphocytes and bone marrow hematopoietic cells in ground squirrels was studied in different seasons and at different stages of the torpor-arousal cycle. The effect of γ-irradiation on animals in different physiological states was also studied. The synthetic activity of cells was estimated from the amount of active RNA per unit DNA in the cell (parameter α). The α values in lymphocytes were minimal in hibernating animals (January–March), reached a peak upon their complete awakening (April), slightly decreased in the summer activity period, and decreased further in the prehibernation autumn period (November). During winter arousals between torpor bouts, this parameter reached the same values as in summer. The dynamics of parameter α in bone marrow hematopoietic cells were generally similar: minimal values in November and higher between torpor bouts than in summer. The peak of synthetic activity of proliferating hematopoietic cells recorded upon awakening from hibernation in April was mainly due to the accumulation of cells in the G₁ and G₂ phases of the cell cycle, and its decrease in summer reflected prevalent transition from G₂ to mitosis and then partly to G₀. In the torpor-arousal-euthermia cycle, two stages of awakening were distinguished, differing considerably in most of the test parameters. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of torpor did not differ significantly from those in nonirradiated torpid animals. The adverse effect of radiation in animals irradiated at the initial stage of awakening was lesser than in animals irradiated in the active state, whereas animals at the second stage of awakening proved more vulnerable to acute irradiation. The physiological state of ground squirrels exposed to ionizing radiation at different phases of the torpor-arousal-euthermia cycle plays a key role in the dynamics of qualitative and quantitative characteristics of blood system cells. The results of this study indicate that the hypometabolic state of ground squirrels during hibernation is a factor of protection from the impact of ionizing radiation on the whole body and on the immune system in particular.     

Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

Prediction of the effect of amino acid substitutions on the thermodynamic stability of proteins is of great importance for studies into the molecular mechanisms underlying the abnormal function of mutant proteins, interpretation of genotyping results, and purposeful design of modified proteins with improved biomedical and biotechnological properties. A set of methods was developed for predicting the changes in free energy (ΔΔG) of mutant proteins containing single substitutions using the information only about protein primary structure or also about the spatial structure. A modified KRAB algorithm was used; its higher accuracy in predicting the changes in the thermodynamic stability of mutant proteins compared with the other known methods designed for solving this problem is demonstrated. Distribution of the positions in the sequence of Malayan pit viper venom protein (kistrin) where the substitutions decrease or increase kistrin stability is analyzed. The substitutions at most positions conserved in the disintegrin family decrease the stability of this protein, except for several positions whose conservation can be determined by functional significance.     

Tracing hybrid incompatibilities to single amino acid substitutions

Deleterious interactions among genes cause reductions in fitness of interpopulation hybrids (hybrid breakdown). Identifying genes involved in hybrid breakdown has proven difficult, and few studies have addressed the molecular basis of this widespread phenomenon. Because proper function of the mitochondrial electron transport system (ETS) requires a coadapted set of nuclear and mitochondrial gene products, ETS genes present an attractive system for studying the evolution of coadapted gene complexes within isolated populations and the loss of fitness in interpopulation hybrids. Here we show the effects of single amino acid substitutions in cytochrome c (CYC) on its functional interaction with another ETS protein, cytochrome c oxidase (COX) in the intertidal copepod Tigriopus californicus. The individual and pairwise consequences of three naturally occurring amino acid substitutions in CYC are examined by site-directed mutagenesis and found to differentially effect the rates of CYC oxidation by COX variants from different source populations. In one case, we show that interpopulation hybrid breakdown in COX activity can be attributed to a single naturally occurring amino acid substitution in CYC.     

Quantifying the effect of burial of amino acid residues on protein stability

The average contribution of individual residue to folding stability and its dependence on buried accessible surface area (ASA) are obtained by two different approaches. One is based on experimental mutation data, and the other uses a new knowledge-based atom-atom potential of mean force. We show that the contribution of a residue has a significant correlation with buried ASA and the regression slopes of 20 amino acid residues (called the buriability) are all positive (pro-burial). The buriability parameter provides a quantitative measure of the driving force for the burial of a residue. The large buriability gap observed between hydrophobic and hydrophilic residues is responsible for the burial of hydrophobic residues in soluble proteins. Possible factors that contribute to the buriability gap are discussed.     

Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.     

Two types of amino acid substitutions in protein evolution

Summary The frequency of amino acid substitutions, relative to the frequency expected by chance, decreases linearly with the increase in physico-chemical differences between amino acid pairs involved in a substitution. This correlation does not apply to abnormal human hemoglobins. Since abnormal hemoglobins mostly reflect the process of mutation rather than selection, the correlation manifest during protein evolution between substitution frequency and physico-chemical difference in amino acids can be attributed to natural selection. Outside of abnormal proteins, the correlation also does not apply to certain regions of proteins characterized by rapid rates of substitution. In these cases again, except for the largest physico-chemical differences between amino acid pairs, the substitution frequencies seem to be independent of the physico-chemical parameters. The limination of the substituents involving the largest physicochemical differences can once more be attributed to natural selection. For smaller physico-chemical differences, natural selection, if it is operating in the polypeptide regions, must be based on parameters other than those examined.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein

Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121-231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121-231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121-231) and similar to full-length PrP(23-231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation. The thermodynamic stabilities of the variants do not correlate with specific disease phenotypes. Five of the amino acid replacements destabilize PrP(121-231), but the other variants have the same stability as the wild-type protein. These data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.