Role of mutational bias and natural selection on genome-wide nucleotide bias in prokaryotic organisms

Correlations between genomic GC contents and amino acid frequencies were studied in the homologous sequences of 12 eubacterial genomes. Results show that amino acids encoded by GC-rich codons increases significantly with genomic GC contents, whereas opposite trend was observed in case of amino acids encoded by GC-poor codons. Further studies show all the amino acids do not change in the predicted direction according to their genomic GC pressure, suggesting that protein evolution is not entirely dictated by their nucleotide frequencies. Amino acid substitution matrix calculated among hydrophobic, amphipathic and hydrophilic amino acid groups' shows that amphipathic and hydrophilic amino acids are more frequently substituted by hydrophobic amino acids than from hydrophobic to hydrophilic or amphipathic amino acids. This indicates that nucleotide bias induces a directional changes in proteome composition in such a way that underwent strong changes in hydropathy values. In fact, significant increases in hydrophobicity values have also been observed with the increase of genomic GC contents. Correlations between GC contents and amino acid compositions in three different predicted protein secondary structures show that hydropathy values increases significantly with GC contents in aperiodic and helix structures whereas strand structure remains insensitive with the genomic GC levels. The relative importance of mutation and selection on the evolution of proteins have been discussed on the basis of these results.     

Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes

Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.     

籽粒苋苹果酸酶基因克隆及分析

NAD/NADP-苹果酸酶(NAD-ME/NADP-ME)是C4植物光合途径的关键酶。采用RT-PCR技术对籽粒苋NAD-ME基因进行克隆,获得了籽粒苋NAD-ME基因的cDNA序列。结果表明,该序列开放可读框长度为1 872 bp,编码623个氨基酸;多序列比对和进化树分析表明,该基因核苷酸序列与其他植物已报道的NAD-ME/NADP-ME基因的核苷酸序列一致性高达75.1%~80.6%,其氨基酸序列与其他植物的NAD-ME/NADP-ME蛋白一致性为73.2%~80.3%。对推断氨基酸序列的蛋白保守区、疏水性/亲水性、潜在跨膜片段、信号肽、蛋白固有无序化和蛋白二级结构分析表明,该蛋白具有苹果酸酶的保守区、兼具亲水性和疏水性,并且含有无序结构域,可能是一种跨膜的非分泌性蛋白。     

A general model of the P2 protein of peripheral nervous system myelin based on secondary structure predictions, tertiary folding principles, and experimental observations

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable alpha-helix, beta-structure, beta-turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high beta-sheet content and virtual absence of alpha-helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the beta-structure over the alpha-helical conformation. In order to define those beta-sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted beta-strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential beta-strands in the predicted secondary structure were then ordered into beta-sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a "Greek key" beta-barrel, consisting of eight antiparallel beta-strands with a two-stranded ribbon of antiparallel beta-structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative beta-strand orderings yield three specific models having different interstrand connections across the barrel ends.     

三联体密码子在mRNA二级结构中的分布

对编码成熟肽的ｍＲＮＡ二级结构的分析显示,每个密码子在ｍＲＮＡ二级结构中的位置有一定的倾向性,这种倾向性似乎与相应氨基酸的构象性质相一致。大多数编码疏水氨基酸的密码子位于ｍＲＮＡ二级结构中较稳定的茎区;反之,大多数编码亲水氨基酸的密码子位于柔性的环区。这个结果支持了最近得到的关于ｍＲＮＡ与蛋白质之间存在丰三维结构信息传递的结论。     

Secondary structures of a new class of lipid body proteins from oilseeds.

The three main isoforms of the 19-kDa lipid body proteins (oleosin) have been purified to homogeneity from embryos of rapeseed. The secondary structures of these proteins as derived from circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy were compared with the secondary structures predicted from the primary sequences. The salient feature of the primary sequence of all oleosins is its division into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains, respectively. Using a variety of predictive methods based on primary amino acid sequence data, the oleosins exhibited a high probability of beta-strand structure in the 70-residue central hydrophobic domain, with relatively little alpha-helical content. Secondary structure data derived from CD and FTIR were consistent with the predictions from primary sequence, showing that the oleosins contained about 45% beta-strand and 13% alpha-helical structure. Under high salt conditions, a 40-kDa polypeptide was obtained from purified preparations of the 19-kDa oleosins. The 40-kDa polypeptide has a very similar secondary structure, as analyzed by CD and FTIR, to that of the 19-kDa oleosins. This polypeptide is therefore probably a dimer of the 19-kDa oleosins that is formed in high salt environments. A model of the general structure of oleosins is proposed whereby the central hydrophobic domain of the protein with a predominantly beta-strand structure is embedded into the non-aqueous phase of lipid-bodies. This hydrophobic region is flanked by putative alpha-helical structures in the polar N- and C-terminal domains which are probably oriented at the lipid-water interface.     

Polarity and hydropathic properties of natural amino acids

A new classification of amino acids according to their polarity and symmetric location in the spatial structure of the genetic code is suggested. The polar amino acids are: R, S (codons AGC and AGU), K, N, Q, H, W, C, Y, G, E, D; apolar ones are: T, M, I, P, L, S (codons UCN). Polar and apolar amino acids are grouped into three families whose members possess complementarity with respect to the symmetric structure of the genetic code. Interaction of these complementary polar and apolar amino acids encodes formation of the space structures and ligand-receptor complexes of proteins. Correlation between the polar and hydropathic properties of amino acids is investigated. Normalization of 38 hydrophobicity scales of natural amino acids is carried out. A discrepancy between structures of polar/hydrophilic and apolar/hydrophobic groups of amino acids is demonstrated. According to the signature principle this discrepancy is due to different properties of amino acid side radicals which, in turn, depend on the second component of the reaction and on environmental conditions.     

Protein evolution within and between species

Protein evolution can be seen as the successive replacement of amino acids by other amino acids. In general, it is a very slow process which is triggered by point mutations in the nucleotide sequence. These mutations can transform into single nucleotide polymorphisms (SNPs) within populations and diverging proteins between species. It is well known that in many cases amino acids can be replaced by others without impeding the functioning of the protein, even if these are of quite different physico-chemical character. In some cases, however, almost any replacement would result in a functionally deficient protein. Based upon comprehensive published SNP data and applying correlation analysis we quantified the two antagonist factors controlling the process of amino acid replacement and thus protein evolution: First, the degenerate structure of the genetic code which facilitates the exchange of certain amino acids and, second, the physico-chemical forces which limit the range of possible exchanges to maintain a functional protein. We found that the observed frequencies of amino acid exchanges within species are best explained by the genetic code and that the conservation of physico-chemical properties plays a subordinate role, but has nevertheless to be considered as a key factor. Between moderately diverged species genetic code and physico-chemical properties exert comparable influence on amino acid exchanges. We furthermore studied amino acid exchanges in more detail for six species (four mammals, one bird, and one insect) and found that the profiles are highly correlated across all examined species despite their large evolutionary divergence of up to 800 million years. The species specific exchange profiles are also correlated to the exchange profile observed between different species. The currently available huge body of SNP data allows to characterize the role of two major shaping forces of protein evolution more quantitatively than before.     

Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.

DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.     

Design and characterization of peptides with amphiphilic beta-strand structures

To extend our studies on peptides and proteins with amphiphilic secondary structures, a series of peptides designed to form amphiphilic beta-strand structures was designed, synthesized, and characterized by circular dichroism and infrared spectroscopy. Amphiphilic beta-strand conformations may be likely to appear in a variety of surface-active proteins, including apolipoprotein B and fibronectin. In a beta-strand conformation, the synthetic peptides will possess a hydrophobic face composed of valine side chains and a hydrophilic face composed of alternating acidic (glutamic acid) and basic (ornithine or lysine) residues. The peptides studied had a variety of chain lengths (5, 9, and 13 residues), and had the amino groups either free or protected with the trifluoroacetyl group. While the peptides did not possess a high potential for beta-sheet formation based on the Chou Fasman parameters, they possessed significant beta-sheet content, with up to 90% beta-sheet calculated for the 13-residue protected peptide. The driving force for beta-sheet formation is the potential amphiphilicity of this conformation. The beta-strand conformation of the 13-residue deprotected peptide was stable in 50% trifluoroethanol, 6 M guanidine hydrochloride, and octanol. The peptides are strongly self-associating in water, which would reduce the unfavorable contacts of the hydrophobic residues with water. It is clear that small peptides can be designed to form stable beta-strand conformations.     

双峰驼凝乳酶原基因的生物信息学分析

通过生物信息学的方法对双峰驼凝乳酶原基因及相应的氨基酸序列的同源性、理化性质、保守结构域、亚细胞定位、信号肽、跨膜结构域、亲水性/疏水性、二级结构进行预测分析.结果表明,双峰驼凝乳酶原基因开放阅读框全长1 146 bp,编码381个氨基酸,属于胃蛋白酶A超家族,预测定位于内质网（膜）的稳定亲水性蛋白,具有一个16个氨基酸的信号肽,其不含跨膜结构域.无规卷曲是其二级结构中最大量的结构元件,α螺旋和延抻链分散于整个蛋白质中,活性位点的分析表明,编码蛋白有6类活性位点.分析双峰驼凝乳酶原基因及其编码蛋白质的特征,能够为深入开展双峰驼凝乳酶的表达和凝乳特性研究提供理论依据.     

Cloning and nucleotide sequence of the chlD locus

The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype.     

Predicted structure of tail-fiber proteins of T-even type phages

The sequences of the tail fiber protein 36 of the phages T4, T2, K3, and Ox2 were analyzed for homologies and for folding patterns using structure prediction methods. No repeating motif was found. A model for the fiber structure is proposed in which beta-strands of about 6 amino acids are separated by turns. In the beta-strand, hydrophobic amino acids are found alternating with hydrophilic ones. Such amphipathic beta-strands can be stabilized by dimer formation. The dimerization occurs in a parallel fashion so that both N-termini are at one end of the dimer. This structure represents a rigid fiber. Our model is consistent with electron microscopic data and electron diffraction patterns for the T4 tail fiber. The observation that all fiber components are found as dimers supports our model. Sequences of the receptor recognition proteins 38 of T-even type phages reveal an architecture different from the architecture of the fiber proteins 36 and 37 of these phages.     

Computational methods for predicting structure of membrane proteins using amino acid sequences

The structure of membrane proteins specifies their functional properties, which are important for medicine and pharmacology and, therefore, is of significant interest. The repetition of transmembrane regions that consist of hydrophobic amino acids is a characteristic and organic feature of polytopic membrane proteins. The ordered repetition (periodicity) can be detected by the Fourier method applied to a digital image of the symbolic amino acid sequence of a protein. In the present work, this investigation was carried out for 24 transmembrane proteins (successfully for 14 of them). If the repetition of transmembrane regions is aperiodic, it can be revealed by another method, that is, the method of the reiterated (four to five times) averaging of the protein hydrophobicity function in a window within the limits of 9–11 amino acids that moves along the sequence. This novel method was applied to the 24 transmembrane proteins (successfully for 19 of them) and demonstrated higher suitability than the Fourier method for predicting the secondary structure of these proteins and the corresponding functional properties.     

解淀粉芽胞杆菌WS-8中LanM基因的克隆及生物信息学分析

克隆解淀粉芽胞杆菌WS-8 (Bacillus amyloliquefaciens WS-8)中的第二类羊毛硫肽合成酶LanM基因,并对LanM编码蛋白的理化性质及结构特征进行分析。设计LanM基因(登录号:APQ49580.1)扩增引物,提取解淀粉芽胞杆菌WS-8基因组DNA,以其作为模板进行PCR扩增。综合多种软件预测和分析LanM编码蛋白的理化性质、结构域和二级结构等。采用邻位连接法(Neighbor-Joining, NJ)构建系统发育树来分析LanM所编码蛋白与同源蛋白的亲缘关系,以及各蛋白结构域的亲缘关系。PCR扩增出的目的条带约为2 840 bp,通过测序鉴定,序列信息与基因组数据一致。LanM基因编码961个氨基酸,等电点为6.07,相对分子质量为111.485 1 kD。LanM编码的蛋白属于亲水性蛋白,无信号肽。该蛋白含有LANC_like结构域,其二级结构主要由螺旋结构和环状结构组成。系统发育树的分析结果表明LANC_like结构域和LanM同源蛋白的亲缘关系一致。解淀粉芽胞杆菌WS-8中存在第二类羊毛硫肽合成酶LanM基因。揭示了羊毛硫肽合成酶LanM发挥脱水和环化作用的理化性质和结构基础,以期为进一步阐明羊毛硫肽合成酶的生物学功能提供参考。     

PSP_MCSVM: brainstorming consensus prediction of protein secondary structures using two-stage multiclass support vector machines

Secondary structure prediction is a crucial task for understanding the variety of protein structures and performed biological functions. Prediction of secondary structures for new proteins using their amino acid sequences is of fundamental importance in bioinformatics. We propose a novel technique to predict protein secondary structures based on position-specific scoring matrices (PSSMs) and physico-chemical properties of amino acids. It is a two stage approach involving multiclass support vector machines (SVMs) as classifiers for three different structural conformations, viz., helix, sheet and coil. In the first stage, PSSMs obtained from PSI-BLAST and five specially selected physicochemical properties of amino acids are fed into SVMs as features for sequence-to-structure prediction. Confidence values for forming helix, sheet and coil that are obtained from the first stage SVM are then used in the second stage SVM for performing structure-to-structure prediction. The two-stage cascaded classifiers (PSP_MCSVM) are trained with proteins from RS126 dataset. The classifiers are finally tested on target proteins of critical assessment of protein structure prediction experiment-9 (CASP9). PSP_MCSVM with brainstorming consensus procedure performs better than the prediction servers like Predator, DSC, SIMPA96, for randomly selected proteins from CASP9 targets. The overall performance is found to be comparable with the current state-of-the art. PSP_MCSVM source code, train-test datasets and supplementary files are available freely in public domain at: and     

An analysis of the helix-to-strand transition between peptides with identical sequence

An analysis of peptide segments with identical sequence but that differ significantly in structure was performed over non-redundant databases of protein structures. We focus on those peptides, which fold into an alpha-helix in one protein but a beta-strand in another. While the study shows that many such structurally ambivalent peptides contain amino acids with a strong helical preference collocated with amino acids with a strong strand preference, the results overwhelmingly indicate that the peptide's environment ultimately dictates its structure. Furthermore, the first naturally occurring structurally ambivalent nonapeptide from evolutionary unrelated proteins is described, highlighting the intrinsic plasticity of peptide sequences. We even find seven proteins that show structural ambivalence under different conditions. Finally, a computer algorithm has been implemented to identify regions in a given sequence where secondary structure prediction programs are likely to make serious mispredictions.     

Asymmetric amino acid compositions of transmembrane beta-strands

In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane beta-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a beta-strand. This new characteristic of beta-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane beta-barrels.     

Carbon - the first frontier of information processing

Information is often encoded as an aperiodic chain of building blocks. Modern digital computers use bits as the building blocks, but in general the choice of building blocks depends on the nature of the information to be encoded. What are the optimal building blocks to encode structural information? This can be analysed by substituting the operations of addition and multiplication of conventional arithmetic with translation and rotation. It is argued that at the molecular level, the best component for encoding discretized structural information is carbon. Living organisms discovered this billions of years ago, and used carbon as the back-bone for constructing proteins that function according to their structure. Structural analysis of polypeptide chains shows that an efficient and versatile structural language of 20 building blocks is needed to implement all the tasks carried out by proteins. Properties of amino acids indicate that the present triplet genetic code was preceded by a more primitive one, coding for 10 amino acids using two nucleotide bases.     

Correlation between amino acid residues converted by RNA editing and functional residues in protein three-dimensional structures in plant organelles

Background

In plant organelles, specific messenger RNAs (mRNAs) are subjected to conversion editing, a process that often converts the first or second nucleotide of a codon and hence the encoded amino acid. No systematic patterns in converted sites were found on mRNAs, and the converted sites rarely encoded residues located at the active sites of proteins. The role and origin of RNA editing in plant organelles remain to be elucidated.

Results

Here we study the relationship between amino acid residues encoded by edited codons and the structural characteristics of these residues within proteins, e.g., in protein-protein interfaces, elements of secondary structure, or protein structural cores. We find that the residues encoded by edited codons are significantly biased toward involvement in helices and protein structural cores. RNA editing can convert codons for hydrophilic to hydrophobic amino acids. Hence, only the edited form of an mRNA can be translated into a polypeptide with helix-preferring and core-forming residues at the appropriate positions, which is often required for a protein to form a functional three-dimensional (3D) structure.

Conclusion

We have performed a novel analysis of the location of residues affected by RNA editing in proteins in plant organelles. This study documents that RNA editing sites are often found in positions important for 3D structure formation. Without RNA editing, protein folding will not occur properly, thus affecting gene expression. We suggest that RNA editing may have conferring evolutionary advantage by acting as a mechanism to reduce susceptibility to DNA damage by allowing the increase in GC content in DNA while maintaining RNA codons essential to encode residues required for protein folding and activity.