Frequency and Markov chain analysis of amino-acid sequences of human glutathione reductase

The amino-acid sequence of human glutathione reductase was measured according to two- and three-amino-acid sequences. The measured frequency and probability were compared with predicted frequency and probability. Of 477 two-amino-acid sequences in human glutathione reductase, 176 (36.897%) and 90 (18.868%) sequences can be explained by the predicted frequency and the predicted probability according to a purely random mechanism. Of 477 measured first Markov transition probabilities for the second amino acid in two-amino-acid sequences, 1 (0.210%) measured first Markov transition probability matches the predicted conditional probability and can therefore be explained by a purely random mechanism. No more than two-amino-acid sequences can be explained by a purely random mechanism.     

Frequency and Markov chain analysis of amino acid sequences of human connective tissue growth factor

The amino acid sequence of human connective tissue growth factor was measured according to two-, three- and four-amino-acid sequences. The measured frequency and probability were compared with predicted frequency and probability. In human connective tissue growth factor, 81 (23.276%) and 21 (6.034%) of 348 two-amino-acid sequences can be explained by the predicted frequency and probability according to a purely random mechanism, 113 (55.122%) and 50 (24.390%) of 205 non-appearing two-amino-acid sequences can be explained by the predicted frequency and probability according to a purely random mechanism; no measured Markov transition probability for the second amino acid in two-amino-acid sequences matches the predicted conditional probability.     

Analysis of distributions of amino acids and amino acid pairs in human tumour necrosis factor precursor and its eight mutations according to a random mechanism

In this study, we use the random principle to analyse the distributions of amino acids and amino acid pairs in human tumour necrosis factor precursor (TNF-!) and its eight mutations, to compare the measured distribution probability with the theoretical distribution probability and to rank the measured distribution probability against the theoretical distribution probability. In this way, we can suggest that distributions with a high random rank should not be deliberately evolved and conserved and those with a low random rank should be deliberately evolved and conserved in human TNF-!. An increased distribution probability in a mutation means probabilistically that the mutation is more likely to occur spontaneously, whereas a decreased distribution probability in a mutation means probabilistically that the mutation is less likely to occur spontaneously and perhaps is more related to a certain cause. The results, for example, show that the distributions of 30% of the amino acids are identical with their probabilistic simplest distributions, and the distributions of some of the remaining amino acids are very close to their probabilistic simplest distributions. With respect to probabilities of distributions of amino acids in mutations, the results show that mutations lead to an increase in eight probabilities, which are thus more likely to occur. Eight probabilities decrease and are thus less likely to occur. With respect to the random ranks against the theoretical probabilities of distributions of amino acids, the results show that mutations lead to an increase in seven and a decrease in seven probabilities, with two probabilities unchanged.     

Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene

The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.     

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

A New Method of Inference of Ancestral Nucleotide and Amino Acid Sequences

A statistical method was developed for reconstructing the nucleotide or amino acid sequences of extinct ancestors, given the phylogeny and sequences of the extant species. A model of nucleotide or amino acid substitution was employed to analyze data of the present-day sequences, and maximum likelihood estimates of parameters such as branch lengths were used to compare the posterior probabilities of assignments of character states (nucleotides or amino acids) to interior nodes of the tree; the assignment having the highest probability was the best reconstruction at the site. The lysozyme c sequences of six mammals were analyzed by using the likelihood and parsimony methods. The new likelihood-based method was found to be superior to the parsimony method. The probability that the amino acids for all interior nodes at a site reconstructed by the new method are correct was calculated to be 0.91, 0.86, and 0.73 for all, variable, and parsimony-informative sites, respectively, whereas the corresponding probabilities for the parsimony method were 0.84, 0.76, and 0.51, respectively. The probability that an amino acid in an ancestral sequence is correctly reconstructed by the likelihood analysis ranged from 91.3 to 98.7% for the four ancestral sequences.     

Proteins as special subsets of polypeptides

A large protein sequence database with over 31,000 sequences and 10 million residues has been analysed. The pair probabilities have been converted to entropies using Boltzmann’s law of statistical thermodynamics. A scoring weight corresponding to “mixing entropy” of the amino acid pairs has been developed from which the entropies of the protein sequences have been calculated. The entropy values of natural sequences are lower than their random counterparts of same length and similar amino acid composition. Based on the results it has been proposed that natural sequences are a special set of polypeptides with additional qualification of biological functionality that can be quantified using the entropy concept as worked out in this paper.     

Multi-modality of pI distribution in whole proteome

Multi-modality of pI distribution is a common feature in different whole proteomes. Some researchers considered it relate to the proteins with different subcellular locations, indicating the result of natural selection. We explored the pI distribution of predicted proteomes (including animals, plants, bacterium, archaeans) and random proteome [random protein sequences constructed according to the special amino acid composition and molecular weight (MW) distribution of human predicted proteome]. Our results suggest that the multi-modality is the result of discrete pK(R) values for different amino acids. Amino acid composition and MW distribution of a proteome also contributes to the specific pI distribution. Although protein subcellular location was related to pI value, our analyses revealed that comparing with the random proteome, neither the multi-modality phenomenon nor the distribution bias of pI values is caused by subcellular location. It seems that the multi-modality distribution is just a mathematical fun. The blank region near the neutral pI was caused by the absence of amino acids with neutral pK(R), and suggests that the selection of amino acids with ionizable side chain might be restricted by the requirement for a special pH environment during the origin of life. From this point of view, the special distribution was the result of natural selection.     

Probing beta-lactamase structure and function using random replacement mutagenesis.

A new analytical mutagenesis technique is described that involves randomizing the DNA sequence of a short stretch of a gene (3-6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein. Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical. We applied this method to 66 codons of the gene encoding TEM-1 beta-lactamase in 19 separate experiments. We found that TEM-1 beta-lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild-type enzyme. We also found a few exceptional regions where only a few random sequences function. Examination of the X-ray structures of homologous beta-lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein. DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related beta-lactamases.     

The uniqueness of protein sequences: A Monte Carlo analysis

Amino acid sequences have already been examined in some detail in order to relate them to structural aspects, homology and gene duplication. This report introduces the concept of internal uniqueness of tripeptides within protein sequences and uses the Monte Carlo method to study this property. Some idea of internal uniqueness may be obtained from such an analysis using only a single sequence if the probability of the random occurrence is about 0.001 or less. This method of analysis is similar to that used in quantitative evaluations of homology. When the probability of the random occurrence is larger than 0.001 a homologous group of sequences is required and the random probabilities may be compared with the real occurrences within the group. From such an examination insulin and cytochrome c are identified as protein sequences with high internal uniqueness. A comparison of data from internal uniqueness and gene duplication analyses shows that these two properties need not be related. Results of the analysis point to internal uniqueness as an additional parameter for inclusion in speculations on why twenty amino acids are coded in protein structure.     

Exact computation of pattern probabilities in random sequences generated by Markov chains

Observed patterns in macromolecular sequences are often consideredas words and compared with their probabilities of occurringin random sequences. Calculation of these probabilities, however,often lacks rigour. We have developed an algorithm for exactcomputation of such probabilities for stochastic sequences thatfollow a Markov chain model. The method is applicable to thecase that a random sequence contains one out of two given patternsP and Q, or both simultaneously. Another application yieldsthe probability Junction P(x) that a sequence contains patternP exactly x times. An application to patterns that include wild-cardcharacters yields probabilities for homonucleotide clustersof a given length. We prove the probability of multiple runsof single nucleotides in the SV40 genome to be in accordancewith the dinucleotide composition of the sequence, althoughit is in conflict with mononucleotide composition. Received on January 10, 1990; accepted on April 23, 1990     

A computational framework to empower probabilistic protein design

MOTIVATION: The task of engineering a protein to perform a target biological function is known as protein design. A commonly used paradigm casts this functional design problem as a structural one, assuming a fixed backbone. In probabilistic protein design, positional amino acid probabilities are used to create a random library of sequences to be simultaneously screened for biological activity. Clearly, certain choices of probability distributions will be more successful in yielding functional sequences. However, since the number of sequences is exponential in protein length, computational optimization of the distribution is difficult. RESULTS: In this paper, we develop a computational framework for probabilistic protein design following the structural paradigm. We formulate the distribution of sequences for a structure using the Boltzmann distribution over their free energies. The corresponding probabilistic graphical model is constructed, and we apply belief propagation (BP) to calculate marginal amino acid probabilities. We test this method on a large structural dataset and demonstrate the superiority of BP over previous methods. Nevertheless, since the results obtained by BP are far from optimal, we thoroughly assess the paradigm using high-quality experimental data. We demonstrate that, for small scale sub-problems, BP attains identical results to those produced by exact inference on the paradigmatic model. However, quantitative analysis shows that the distributions predicted significantly differ from the experimental data. These findings, along with the excellent performance we observed using BP on the smaller problems, suggest potential shortcomings of the paradigm. We conclude with a discussion of how it may be improved in the future.     

猪胸膜肺炎放线杆菌转铁结合蛋白基因（rbp8）分析及建模

为了深入研究猪胸膜肺炎放线杆菌（Actinobacillus pleuropneumonie,App）转铁结合蛋白基因（Transferrin BindingProtein8,脚）的生物学特性,采用生物信息学方法,对GenBank中的5株App的TbpB的核酸及其氨基酸序列进行比对,选取其中的中国湖北分离株（JL03）对其分子结构、理化性质及功能域、蛋白质二级和三级结构等重要参数进行了预测和分析,并在三级结构的基础上进行了同源建模。结果表明,不同APP菌株之间核酸序列相似性较大,而氨基酸序列存在较大差异,二级结构以延伸链和随机卷曲为主要构件,其空间结构与脑膜炎双球菌GNAl870蛋白相似性较高,以此为模板成功构建了三维结构分子模型,为TbpB基因功能的深入研究提供了线索和参考依据。     

Mutants generated by the insertion of random oligonucleotides into the active site of the beta-lactamase gene

We have remodeled the gene coding for beta-lactamase by replacing DNA at the active site with random nucleotide sequences. The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 x 10(6) amino acid substitutions. From a population of Escherichia coli infected with plasmids containing these random inserts, we have selected seven new active-site mutants that render E. coli resistant to carbenicillin and a series of related analogues. Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70. Each of the mutants exhibits a temperature-sensitive beta-lactamase activity. This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants.     

Close evolutionary relationship between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase gene mediated by R plasmids

Sixty-three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds of beta-lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe.     

Nonrandom amino acid substitution and estimation of the number of nucleotide substitutions in evolution

Summary A method of estimating the number of nucleotide substitutions from amino acid sequence data is developed by using Dayhoff's mutation probability matrix. This method takes into account the effect of nonrandom amino acid substitutions and gives an estimate which is similar to the value obtained by Fitch's counting method, but larger than the estimate obtained under the assumption of random substitutions (Jukes and Cantor's formula). Computer simulations based on Dayhoff's mutation probability matrix have suggested that Jukes and Holmquist's method of estimating the number of nucleotide substitutions gives an overestimate when amino acid substitution is not random and the variance of the estimate is generally very large. It is also shown that when the number of nucleotide substitutions is small, this method tends to give an overestimate even when amino acid substitution is purely at random.     

Determination of mutation trend in proteins by means of translation probability between RNA codes and mutated amino acids

In this study, we estimate the translation probability to amino acid from RNA codon. With the determined 183 translation probabilities and amino-acid composition of eight highly mutated proteins, we construct the theoretical distributions of mutated amino acids in these proteins and then compare them with their actual distributions affected by mutations. Thereafter we trace the pattern of translation probabilities from RNA codons to mutated amino acids of 1053 point missense mutations. Finally, we statistically conclude that the natural mutation trend goes along the theoretical translation probability.     

Characterization of an extended-spectrum class C beta-lactamase of Citrobacter freundii

Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino beta-lactams such as ceftazidime and aztreonam. This drug resistance was due to an extended-spectrum class C beta-lactamase encoded by chromosomal gene(s). The GC3 beta-lactamase showed high amino acid sequence homology to a known C. freundii beta-lactamase, i.e., 346 of 361 amino acids were identical with those of C. freundii GN346 beta-lactamase (Tsukamoto, K. et al, Eur. J. Biochem. 188, 15-22, 1990). Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended-spectrum resistance in class C beta-lactamases (Haruta, S. et al, Microbiol. Immunol. 42, 165-169, 1998). However, Asp198 was eliminated as a cause of the extended-spectrum resistance by the substitution of Asn for Asp198. Subsequent investigation suggested that the moderate resistance to oxyimino beta-lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active-site. Some or all of the replacements are assumed to delicately modify the active-site configuration. The GC3 beta-lactamase is the first example of an extended-spectrum class C beta-lactamase in which mutations are independent of the omega loop.