Code dependent conservation of the physicochemical 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.This work was partially supported by OEA and Departamento de Desarrollo de la Investigación.     

Analysis of nucleotide substitutions and amino acid conservation in theDrosophila Adh genomic region

The homologous genomic region that contains two paralogous genes,Adh andAdh-dup, was compared in severalDrosophila species. Sequences were analyzed as follows: a) At the nucleotide level, Ka and Ks values were determined for each pair of species. Ka-Adh and Ka-Adh-dup are not significantly different. However, Ks-Adh values are significantly lower than Ks-Adh-dup, which are more variable. In agreement with other reports, lower Ks values forAdh correlate with a high level of gene expression and relatively high percentage of G+C content in the third codon position, while the opposite applies toAdh-dup. b) At the protein level, amino acid comparisons reveal conserved regions shared by ADH and ADH-DUP, which have been assigned to known functional domains. Key residues for dehydrogenasic function are also found in ADH-DUP, thus pointing to a dehydrogenase activity for ADH-DUP, albeit very different from that of ADH.     

Conservation of physico-chemical amino acid properties during the evolution of proteins

Based on a similarity ring constructed from a substitution probability matrix, we have analyzed the conservation of some amino acid properties in the evolution of proteins. Refractive index and bulkiness are highly conserved, hydrophobicity and polarity are fairly well conserved while optical rotation appears to be a less relevant property. On the other hand, the analysis of the correspondence between phenotype and genotype shows that the most frequent amino acid substitutions in proteins do not always correspond to the most feasible codon changes. The apparent disagreement between amino acid substitutions in modern proteins and the primordial amino acid-codon assignment is discussed.     

Single amino acid substitutions in puroindoline-b mutants influence lipid binding properties

External reflectance Fourier transform infrared (ER-FTIR) spectroscopy and surface pressure measurements have been used to characterize the interaction of wild-type puroindoline-b (Pin-b) and two mutant forms featuring single residue substitutions-namely, Gly-46 to Ser-46 (Pin-bH) and Trp-44 to Arg-44 (Pin-bS)-with condensed-phase monolayers of zwitterionic (L-alpha-dipalmitoylphosphatidylcholine, DPPC) and anionic (L-alpha-dipalmitoylphosphatidyl-dl-glycerol, DPPG) phospholipids. The interaction with anionic DPPG monolayers, monitored by surface pressure isotherms, was influenced significantly by mutations in Pin-b (p < 0.05); wild-type Pin-b showed the highest surface pressure change of 10.6 +/- 1.0 mN m-1, followed by Pin-bH (7.9 +/- 1.6 mN m-1) and Pin-bS (6.3 +/- 1.0 mN m-1), and the surface pressure isotherm kinetics were also different in each case. Integrated Amide I peak areas from corresponding ER-FTIR spectra confirmed the differences in adsorption kinetics, but also showed that differences in adsorbed amount were less significant, suggesting that mutations influence the degree of penetration into DPPG films. All Pin-b types showed evidence of interaction with DPPC films, detected as changes in surface pressure (5.6 +/- 1.1 mN m-1); however, no protein peaks were detected in the ER-FTIR spectra, which indicated that the interaction was via penetration with limited adsorption at the lipid/water interface. The expression of Pin-b mutants is linked to wheat endosperm hardness; therefore, the data presented here suggest that the lipid binding properties may be pivotal within the mechanism for this quality trait. In addition, the data suggest antimicrobial activities of Pin-b mutants would be lower than those of the wild-type Pin-b, because of decreased selectivity toward anionic phospholipids.     

Combinatorial codon-based amino acid substitutions

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 β-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the β-lactamase omega loop region.     

The regularities of the changes of amino acid physico-chemical properties within the genetic code

Summary All the codons of the genetic code can be arranged into the closed one-step mutation ring, containing three periods of the same sequence of mutations (2,3,3,3,1,3,3,3,1,3,3,3,1,3,3,3,2,3,3,3). The codons of Gly play a role of the connecting element between the end of the third, and the beginning of the first period of the genetic code. The reactivity of amino acids, expressed by the reaction rates of aminolysis reaction of N-hydroxysuccinimide esters of protected amino acids with p-anisidine, changes periodically with the respect to the mutation periods of the genetic code. Chou-Fasman P as well as P conformational parameters of amino acids, and also the compositional frequencies of amino acids in proteins, demonstrate the pseudosymmetry pattern with respect to the center of one-step mutation ring, which is situated between Thr ACY and ACR codons.     

Selection on amino acid substitutions in Arabidopsis

Studies of nucleotide diversity have found an excess of low-frequency amino acid polymorphisms segregating in Arabidopsis thaliana, suggesting a predominance of weak purifying selection acting on amino acid polymorphism in this inbreeding species. Here, we investigate levels of diversity and divergence at synonymous and nonsynonymous sites in 6 circumpolar populations of the outbreeding Arabidopsis lyrata and compare these results with A. thaliana, to test for differences in mutation and selection parameters across genes, populations, and species. We find that A. lyrata shows an excess of low-frequency nonsynonymous polymorphisms both within populations and species wide, consistent with weak purifying selection similar to the patterns observed in A. thaliana. Furthermore, nonsynonymous polymorphisms tend to be more restricted in their population distribution in A. lyrata, consistent with purifying selection preventing their geographic spread. Highly expressed genes show a reduced ratio of amino acid to synonymous change for both polymorphism and fixed differences, suggesting a general pattern of stronger purifying selection on high-expression proteins.     

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.     

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.     

Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions.

The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.     

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.     

How do amino acid substitutions affect the amyloidogenic properties and seeding efficiency of prion peptides

The amino acid sequences in the amyloidogenic region (amino acids 108–144) of several mammalian prion proteins were compared and variations were found to occur at residues 109 (M or L), 112 (M or V), 129 (M, V, or L), 135 (N or S), 138 (M, L, or I), 139 (M or I), and 143 (N or S). Using the bovine PrP peptide (residues 108–144 based on the numbering of the human prion protein sequence) as a control peptide, several peptides with one amino acid differing from that of the bovine PrP peptide at residues 109, 112, 135, 138, 139, or 143 and several mammalian PrP peptides were synthesized, and the effects of these amino acid substitutions on the amyloidogenic properties of these peptides were compared and discussed on the basis of the chemical and structural properties of amino acids. Our results showed that the V112M substitution accelerated nucleation of amyloidogenesis, while the N143S and I139M substitutions retarded nucleation. These effects tended to cancel each other out when two substitutions with opposite effects were present on the same peptide. Moreover, acceleration or inhibition of nucleation was not necessarily correlated with effect on seeding efficiency. Using amyloid fibrils prepared from the bovine PrP peptide as seeds, the seeding efficiency for the monomer peptides with the M129L, S135N, N143S, or I139M substitution was decreased compared to that for bPrP peptide. Of all the mammalian peptides used in this study, the dog, mule deer, and pig PrP peptides had the lowest seeding efficiencies.     

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/.     

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.     

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.     

Nature of amino acid substitutions in homologous proteins during evolution

Replacement substitutions of mitochondrial cytochrome $\text{c}$ and α- and β-chains of haemoglobin have been studied by considering the structural similarity among amino acid residues at the secondary and tertiary structural levels. Secondary structural similarity explains ～70% while tertiary structural similarity explains ～50% of observed replacements for most of the cases. These structural similarities could not account for all the replacement substitutions. The study was extended to consider the composition of codons, and the chemical nature and polarity of the replacing and replaced residues. These also could not individually account for all the affected replacements. In general, no property of amino acid residues is conserved for substitutions occurring at any single position during evolution of proteins.