HIV-1 protease catalytic efficiency effects caused by random single amino acid substitutions

Protein evolution has occurred by successive fixation of individual mutations. The probability of fixation depends on the fitness of the mutation, and the arising variant can be deleterious, neutral, or beneficial. Despite its relevance, only few studies have estimated the distribution of fitness effects caused by random single mutations on protein function. The human immunodeficiency virus type 1 (HIV-1) protease was chosen as a model protein to quantify protein's tolerability to random single mutations. After determining the enzymatic activity of 107 single random mutants, we found that 86% of single mutations were deleterious for the enzyme catalytic efficiency and 54% lethal. Only 2% of the mutations significantly increased the catalytic efficiency of the enzyme. These data demonstrate the vulnerability of HIV-1 protease to single random mutations. When a second random mutagenesis library was constructed from an HIV-1 protease carrying a highly deleterious single mutation (D30N), a higher proportion of mutations with neutral or beneficial effect were found, 26% and 9%, respectively. Importantly, antagonist epistasis was observed between deleterious mutations. In particular, the mutation N88D, lethal for the wild-type protease, restored the wild-type catalytic efficiency when combined with the highly deleterious mutation D30N. The low tolerability to single random substitutions shown here for the wild-type HIV-1 protease contrasts with its in vivo ability to generate an adaptive variation. Thus, the antagonist epistasis between deleterious or lethal mutations may be responsible for increasing the protein mutational robustness and evolvability.     

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.     

Folding defects caused by single amino acid substitutions in a subunit are not alleviated by assembly

Significant stabilization of a protein often occurs when it is assembled into an oligomer. Bacteriophage P22 contains 420 monomers of coat protein that are stabilized by the assembly and maturation processes. The effects of eight single amino acid substitutions in coat protein that each cause a temperature-sensitive-folding defect were investigated to determine if the conformational differences previously observed in the monomers could be alleviated by assembly or maturation. Several techniques including differential scanning calorimetry, heat-induced expansion, urea denaturation, and sensitivity to protease digestion were used to explore the effects of the amino acid substitutions on the conformation of coat protein, once assembled. Each of the amino acid substitutions caused a change in the conformation as compared to wild-type coat protein, observed by at least one of the probes used. Thus, neither assembly nor expansion entirely corrected the conformational defects in the monomeric subunits of the folding mutants.     

Enhancing antimicrobial activity of mastoparan-B by amino acid substitutions

This study evaluated antimicrobial and hemolytic activities of mastoparan-B (MP-B) isolated from the venom of the hornet, Vespa basalis, and its analogs after substituting certain amino acid (aa) residues. MP-B exhibited significantly different antimicrobial activities against bacteria species/strains tested, especially two Escherichia coli strains, Staphylococcus xylosus and Citrobacter koseri at low dosages, and was non-specific against certain Gram-positive and -negative bacteria. Our results indicated that hydrophobicity modification by single aa substitution may enhance their antimicrobial activities. An aa substituted MP-B, viz., MP-B-1, in which Trp substituted for Leu³, became more effective than the original peptide at inhibiting or killing the bacterial species tested, especially Klebsiella pneumonia, Salmonella typhimurium, and Salmonella Cholerasuis, and even up to 8 times more effective in some cases. However, MP-B-2 was virtually similar to MP-B against each bacterial species assayed, while MP-B-3 reduced its effectiveness greatly compared to others. On the other hand, MP-B and its analogs were not effective against the beneficial probiotics and they were not hemolytic to erythrocytes at the dosages tested. Our results suggested that MP-B becomes more potent against specific pathogenic bacteria and safe to the probiotics after undergoing appropriate amino acid substitutions.     

Conformational effects of amino acid substitutions in the P-glycoprotein of themdr 1 gene

The P-glycoprotein of themdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells. The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185. A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchichine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin. This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution. Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185. The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energetically unfavorable for the protein withl-amino acids (including valine) at this position. This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins.     

Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide.

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted alpha-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 +/- 4% alpha-helical structure at 22 degrees C that melts reversibly with T(m,WT) = 50 +/- 2 degrees C and van't Hoff enthalpy deltaH(v,WT)(Tm) = 18 +/- 2 kcal/mol. G15A substitution leads to an increased alpha-helical content of 42 +/- 4% and an increased T(m,G15A) = 57 +/- 2 degrees C, which corresponds to stabilization by delta deltaG(app) = +0.4 +/- 1.5 kcal/mol. G15P mutant has approximately 20% alpha-helical content at 22 degrees C and unfolds with low cooperativity upon heating to 90 degrees C. R23P and T45P mutants are fully unfolded at 0-90 degrees C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative alpha-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal alpha-helical content reaching 74%. Comparison with the maximal alpha-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.     

Reciprocal amino acid substitutions in the evolution of homologous peptides

Amino acid sequences of peptides are often inferred from their amino acid compositions by comparison with homologous peptides of known sequence. The probabilities are considered that by such an approach errors are made due to the occurrence of balanced double changes, i.e. reciprocal substitutions, between two homologous peptides of identical compositions. Formulae are derived for the calculation of these probabilities, depending on peptide length and evolutionary distance. However, such calculations requiring too much computer time, the probabilities for reciprocal substitutions are estimated by simulation of evolutionary changes in peptides. It can be concluded from the resulting data that for many purposes the possible errors in amino acid sequences partially inferred from amino acid compositions are acceptably small.     

Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function

Scavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis.     

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.     

Probing intein-catalyzed thioester formation by unnatural amino acid substitutions in the active site

Inteins are single-turnover catalysts that splice themselves out of a precursor polypeptide chain. For most inteins, the first step of protein splicing is the formation of a thioester through an N-S acyl shift at the upstream splice junction. However, the mechanism by which this reaction is achieved and the impact of mutations in and close to the active site remain unclear on the atomic level. To investigate these questions, we have further explored a split variant of the Ssp DnaB intein by introducing substitutions with unnatural amino acids within the short synthetic N-terminal fragment. A previously reported collapse of the oxythiazolidine anion intermediate into a thiazoline ring was found to be specificially dependent on the methyl side chain of the flanking Ala(-1). The stereoisomer d-Ala and the constitutional isomers β-Ala and sarcosine did not lead to this side reaction but rather supported splicing. Substitution of the catalytic Cys1 with homocysteine strongly inhibited protein splicing; however, thioester formation was not impaired. These results argue against the requirement of a base to deprotonate the catalytic thiol group prior to the N-S acyl shift, because it should be misaligned for optimal proton abstraction. A previously described mutant intein evolved for more general splicing in different sequence contexts could even rather efficiently splice with this homocysteine. Our findings show the large impact of some subtle structural changes on the protein splicing pathway, but also the remarkable tolerance toward other changes. Such insights will also be important for the biotechnological exploitation of inteins.     

Uneven distribution of amino acid substitutions throughout the amino acid sequence of homologous proteins]

A set of aligned homologous protein sequences is divided into two groups consisting of m and n most related sequences. The value of position variability for homologous protein sequences is defined as a number of failures to coincide in the intergroup comparison of all possible m*n pairs of amino acid residues in that position divided by m*n. The position variability value plotted versus the sequence position number with a window of 10 positions gives the intergroup local variability profile. Area S of the figure included between the local variability profile and the straight line corresponding to the mean local variability value is compared with the average area Sr for 1000 random homologous protein families. If S is greater than Sr by more than 2 standard deviation units sigma r, the local variability profile is assumed to contain peaks and hollows corresponding to significant variable and conservative regions of the sequences. The profile extrema containing the area surplus delta S = S-(Sr+ 2 sigma r) are cut off by two straight lines to locate significant regions. The difference (S-Sr) given in standard deviation units sigma r is believed to be the amino acid substitution overall irregularity along the homologous protein sequences OI = (S-Sr)/sigma r. The significant conservative and variable regions of six homologous sequence families (phospholipase A2, cytochromes b, alpha-subunits of Na,K-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre and human rhodopsins) were identified. It was shown that for artificial homologous protein sequences derived by k-fold lengthening of natural protein sequences, the OI value rises as square root of k. To compare the degree of substitution irregularity in homologous protein sequence families of different lengths L the value of standard substitution overall irregularity for L = 250 is proposed.     

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.     

Correlation of co-ordinated amino acid substitutions with function in viruses related to tobacco mosaic virus

Sequence data are available for the coat proteins of seven tobamoviruses, with homologies ranging from at least 26% to 82%, and atomic co-ordinates are known for tobacco mosaic virus (TMV) vulgare. A significant spatial relationship has been found between groups of residues with identical amino acid substitution patterns. This strongly suggest that their location is linked to a particular function, at least in viruses identical with the wild-type for these residues. The most conserved feature of TMV is the RNA binding region. Core residues are conserved in all viruses or show mutations complementary in volume. The specificity of inter-subunit contacts is achieved in different ways in the three more distantly related viruses.     

Code dependent conservation of the physico-chemical properties in amino acid substitutions

The frequency of amino acid replacements in families of typical proteins has been elegantly analyzed by Argyle (1980) showing that the most frequent replacements involve a conservation of the amino acid chemical properties. The cyclic arrangement of the twenty amino acids resulting from the most frequent replacements has been described as an amino acid chemical ring. In this work, a novel amino acid replacement frequency ring is proposed, for which a conservation of over 90% of the most general physico-chemical properties can be deduced. The amino acid chemical similarity ring is also analyzed in terms of the genetic code base probability changes, showing that the discrepancy that exists between the standard deviation value of the amino acid replacement frequency matrix and its respective ideal value is almost equal to that deduced from the corresponding base codon replacement probability matrices. These differences are finally evaluated and discussed in terms of the restrictions imposed by the structure of the genetic code and the physico-chemical dissimilarities between some codons of amino acids which are chemically similar.     

Effects of single-point amino acid substitutions on the structure and function neuraminidase proteins in influenza A virus

In order to clarify the effect of amino acid substitutions on the structure and function of the neuraminidase (NA) protein of influenza A virus, we introduced single-point amino acid substitutions into the NA protein of the A/Tokyo/3/67 (H2N2) strain using PCR-based random mutation. The rate of tolerant random one amino acid substitutions in the NA protein was 47%. Rates of tolerant substitutions for the stalk and for the surface and inner portion of the head region of the NA protein were 79, 54, and 19%, respectively. Deleterious changes, such as those causing the NA protein to stop at the Golgi/endoplasmic reticulum, were scattered throughout the protein. On the other hand, the ratio of mutations with which the NA protein lost neuraminidase activity, but was transported to the cell surface, decreased in proportion to the distance from the structural center of enzyme active site. In order to investigate the effect of accumulated amino acid substitutions on the structural character of the N2NA protein during evolution, the same amino acid substitutions were introduced by site-directed mutagenesis at 23 homologous positions on N2 proteins of A/Tokyo/3/67, A/Bangkok/15/85 (H3N2), and A/Mie/1/2004 (H3N2). The results showed a shift, or discordance, in tolerance at some of the positions. An increase in discordance was correlated with the interval in years between virus strains, and the discordance rate was estimated to be 0.6-0.7% per year.