Determination of amino acid pairs sensitive to variants in human beta-glucocerebrosidase by means of a random approach

In this data-based theoretical analysis, we use the random approach to analyse the amino acid pairs in human beta-glucocerebrosidase in order to determine which amino acid pairs are more sensitive to 109 variants from missense mutant human glucocerebrosidase. The rationale of this study is based on our hypothesis and findings that the harmful variants are more likely to occur at randomly unpredictable amino acid pairs and the non-harmful variants are more likely to occur at randomly predictable amino acid pairs. This is because we argue that the randomly predictable amino acid pairs should not be deliberately evolved, whereas the randomly unpredictable amino acid pairs should be deliberately evolved with connection of protein function. The results show, for example, that 93.58% of 109 variants occur at randomly unpredictable amino acid pairs, which account for 71.40% of amino acid pairs in glucocerebrosidase, and the chance of occurrence of the variant is about 4.4 times higher in randomly unpredictable amino acid pairs than in predictable pairs. Hence the randomly unpredictable amino acid pairs are more sensitive to variants in human glucocerebrosidase. The results also suggest that human glucocerebrosidase has a natural tendency to variants.     

Determination of amino acid pairs sensitive to variants in human copper-transporting ATPase 2

In this study, we use our probabilistic approach to analyze the amino acid pairs in human copper-transporting ATPase 2 (ATP7B) in order to determine which amino acid pairs are more sensitive to 125 variants from missense mutant human ATP7B. The results show 97.6% of 125 variants occur at randomly unpredictable amino acid pairs, which account for 80.9% of amino acid pairs in ATP7B, and the chance of occurring of variant is about 9 times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, the randomly unpredictable amino acid pairs are more sensitive to variants in human ATP7B.     

Determination of amino acid pairs in human p53 protein sensitive to mutations/variants by means of a random approach

This is the continuation of our studies using random approaches to analyse the p53 protein family. In this data-based theoretical analysis, we use the random approach to analyse the amino acid pairs in human p53 protein in order to determine which amino acid pairs are more sensitive to 190 human p53 mutations/variants. The rationale of this study is based on our hypothesis and findings that a harmful mutation is more likely to occur at randomly unpredictable amino acid pairs, and a harmless mutation is more likely to occur at randomly predictable amino acid pairs. This is because we argue that the randomly predictable amino acid pairs should not be deliberately evolved, whereas the randomly unpredictable amino acid pairs should be deliberately evolved with a connection to protein function. The results show, for example, that 93.16% of 190 mutations/variants occur at randomly unpredictable amino acid pairs. Thus, the randomly unpredictable amino acid pairs are more sensitive to mutations/variants in human p53 protein. The results also suggest that the human p53 protein has a tendency for the occurrence of mutation/variants.     

Estimation of amino acid pairs sensitive to variants in human phenylalanine hydroxylase protein by means of a random approach

In this data-based theoretical analysis, we use a random approach to estimate amino acid pairs in human phenylalanine 4-hydroxylase (PAH) protein in order to determine which amino acid pairs are more sensitive to 187 variants in human PAH protein. The rationale of this study is based on our hypothesis and previous findings that the harmful variants are more likely to occur at randomly unpredictable amino acid pairs rather than at randomly predictable pairs. This is reasonable to argue as randomly predictable amino acid pairs are less likely to be deliberately evolved, whereas randomly unpredictable amino acid pairs are probably deliberately evolved in connection with protein function. 94.12% of 187 variants occurred at randomly unpredictable amino acid pairs, which accounted for 71.84% of 451 amino acid pairs in human PAH protein. The chance of a variant occurring is five times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, randomly unpredictable amino acid pairs are more sensitive to variance in human PAH protein. The results also suggest that the human PAH protein has a natural tendency towards variants.     

Analysis of amino acid pairs sensitive to variants in human collagen alpha5(IV) chain precursor by means of a random approach

In this data-based theoretical analysis, we use the random approach to analyze the amino acid pairs in 5(IV) chain precursor (CA54) in order to determine which amino acid pairs are more sensitive to 151 variants from missense mutant human CA54 protein. The rationale of this study is based on our hypothesis and previous findings that harmful variance is more likely to occur at randomly unpredictable amino acid pair position rather than at randomly predictable positions. This is reasonable to argue as randomly predictable amino acid pairs are less likely to be deliberately evolved, whereas randomly unpredictable amino acid pairs are probably deliberately evolved in connection with protein function. The results show that all 151 variants occurred at randomly unpredictable amino acid pairs and the chance of a variant occurring is markedly higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, randomly unpredictable amino acid pairs are more sensitive to variance in human CA54. The results also suggest that the human CA54 protein has a natural tendency towards variants.     

Amino acid pairs susceptible to variants in human protein C precursor

In this study, we analyze the amino acid pairs in human protein C precursor to determine which amino acid pairs are more susceptible to 71 variants from missense mutant human protein C precursor. The results show 85.92% of 71 variants occur at randomly unpredictable amino acid pairs accounting for 61.96% of amino acid pairs in protein C.     

Determination of sensitive positions to mutations in human p53 protein

Over last several years, we demonstrated that the mutations are more likely to occur at randomly unpredictable amino acid pairs in a protein. We therefore can in principle predict the amino acid pairs sensitive to the future mutations in a protein. However, we still need to predict the positions at which the sensitive amino acid pairs are located in a protein. In this study, we use a probabilistic approach to analyze the effect of 191 mutations in human p53 protein and can approximately estimate the sensitive positions to mutations in human p53 protein.     

Prediction of amino acid pairs sensitive to mutations in the spike protein from SARS related coronavirus

In this study, we analyzed the amino acid pairs affected by mutations in two spike proteins from human coronavirus strains 229E and OC43 by means of random analysis in order to gain some insight into the possible mutations in the spike protein from SARS-CoV. The results demonstrate that the randomly unpredictable amino acid pairs are more sensitive to the mutations. The larger is the difference between actual and predicted frequencies, the higher is the chance of mutation occurring. The effect induced by mutations is to reduce the difference between actual and predicted frequencies. The amino acid pairs whose actual frequencies are larger than their predicted frequencies are more likely to be targeted by mutations, whereas the amino acid pairs whose actual frequencies are smaller than their predicted frequencies are more likely to be formed after mutations. These findings are identical to our several recent studies, i.e. the mutations represent a process of degeneration inducing human diseases.     

Low level sequence variant analysis of recombinant proteins: an optimized approach

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.     

Amino Acid Changes in Disease-Associated Variants Differ Radically from Variants Observed in the 1000 Genomes Project Dataset

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.     

Advantages to mutagenesis techniques generating populations containing the complete spectrum of single codon changes

The limitations of current mutagenesis techniques are analyzed in terms of the number and kinds of codon changes they make and in terms of the population size needed to produce all single or multiple amino acid variants. It is shown how a technique that can alter a single codon of a gene, producing all possible variant codons without affecting the rest of the gene, has certain advantages, if it can be used at each place in the gene in one experiment. Such a technique has advantages when the goals are to understand: (1) how specific structural alterations in a mutant protein cause it to function in a different but specific way, (2) how to predict which amino acids in a protein contact or interact with each other, and (3) why a protein is more or less sensitive to mutational disruption, depending upon the specific mutation. This is because it would generate the maximum number of (1) mutant proteins with different functions, (2) intracistronic suppressor for any starting mutation, and (3) random amino acid substitutions at random places. Furthermore, such a technique could produce useful variants more quickly and on a smaller scale than either evolution or current methods.     

Two commonly occurring nucleotide base substitutions in Chinese G6PD variants

Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.     

Regularities in the primary structure of proteins

In this paper the latest protein database consisting of more than a million amino acids is analyzed to characterize the short range regularities in the primary structure. The amino acid distributions along the polypeptide chain and among the proteins have been studied first. Their influence on the amino acid pair statistics was taken into account. We are primarily interested in the distances of the covalent structure, where the amino acid pair frequencies show non-random characters. The amino acid pairs separated by at least 20 residues in the covalent structure exhibit an exact Gaussian distribution. We found that there is a range of non-random pairing in the covalent structure. We conclude that the pair preference characters are different for each of the 20 x 20 amino acid pairs. The range of the non-random pairing varies from pair to pair, and in most cases it does not extend beyond the 9th neighbour. The preferences of a certain pair in a certain position can not be derived from the character of that pair in another position. The preference values of 400 amino acid pairs are listed for up to the pairs in 9th neighbour position. Some fields of potential application of these data have also been discussed.     

Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.     

Functional significance of conserved amino acid residues.

A systemic study of single amino acid substitutions in bacteriophage T4 lysozyme permitted a test of the concept that conserved amino acid residues are more functionally important than nonconserved residues. Substitutions of amino acid residues that are conserved among five bacteriophage-encoded lysozymes were found to lead more frequently to loss of function than substitutions of nonconserved residues. Of 163 residues tested, only 74 (45%) are sensitive to at least one substitution; however, all 14 residues that are fully conserved are sensitive to substitutions.     

Analysis of demethylaminoazobenzene-thiohydantoins of amino acid by high-pressure liquid chromatography

Dimethylaminoazobenzene-thiohydantoins of amino acid can be quantitatively analyzed by high-pressure liquid chromatography at picomole level. As little as 5 to 10 pmol of dimethylaminoazobenzene-thiohydantoins of amino acid can easily be detected in the visible region (436 nm) against a stable baseline. Three amino acid pairs, namely glutamine and threonine, methionine and proline, and leucine and isoleucine, have not yet been separated. This new technique provides a sensitive and efficient tool for measuring the recovery of amino terminal amino acids using the dimethylaminoazobenzene-isothiocyanate method and the repetitive yield of sequence determination using the dimethylaminoazobenzene-isothiocyanate phenylisothiocyanate double-coupling method.     

Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants

Naturally occurring human immunodeficiency virus (HIV-1) variants require the presence of CD4 and specific chemokine receptors to enter a cell. In the laboratory, HIV-1 variants that are capable of bypassing CD4 and utilizing only the CCR5 chemokine receptor for virus entry have been generated. Here we report that these CD4-independent viruses are significantly more sensitive to neutralization by soluble CD4 and a variety of antibodies. The same amino acid changes in the HIV-1 gp120 envelope glycoprotein determined CD4 independence and neutralization sensitivity. The CD4-independent envelope glycoproteins exhibited higher affinity for antibodies against CD4-induced gp120 epitopes but not other neutralizing ligands. The CD4-independent envelope glycoproteins did not exhibit increased lability relative to the wild-type envelope glycoproteins. The utilization of two receptors apparently allows HIV-1 to maintain a more neutralization-resistant state prior to engaging CD4 on the target cell, explaining the rarity of CD4 independence in wild-type HIV-1.     

Geometric preferences of crosslinked protein-derived cofactors reveal a high propensity for near-sequence pairs

Protein-derived cofactors that are composed of covalently crosslinked amino acid side chains are of increasing importance in protein science. These crosslinked protein-derived cofactors (CPDC) are formed either through direct oxidation by metal/O(2)-derived intermediates or through outer sphere oxidation by highly oxidizing cofactors. CPDCs that are formed by outer sphere oxidation do not require side-chain precursors to be coordinated by a metal center, and therefore are more difficult to identify than those formed by direct oxidation. To better understand the propensity for CPDC formation by outer sphere oxidation, the geometrical preferences of CPDCs were examined. The Dezymer algorithm has been used to identify all putative CPDC-forming mutations in 500 proteins. Geometrically, although chemically unrelated, these CPDCs were found to be similar to disulfide-bonded cysteine pairs. Additionally, the percentage of near-sequence pairs (i and i +1 to i and i + 5) increased as the average C(alpha)-C(alpha) distance between the amino acid pairs increased. This survey also examined the protein databank for proteins with pre-attack conformations for CPDCs, using non-bonded contacts reported by Procheck. A total of 323 unique proteins was identified, with 55 being near-sequence amino acid pairs. The high geometric propensity of near-sequence amino acid pairs for forming CPDCs is significant due to difficulties associated with detection by structural or mass spectrometric methods.     

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.     

Function of the N-terminal segment of the RecA-dependent nuclease Ref

The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.