Tracing specific synonymous codon-secondary structure correlations through evolution

Abstract We previously showed that GAU codons are preferred (relative to synonymous GAC codons) for encoding aspartates specifically at the N-termini of α-helices in human, but not in E. coli, proteins. To test if this difference reflected a general difference between eucaryotes and procaryotes, we now extended the analysis to include the proteins and coding sequences of mammals, vertebrates, S. cerevisiae, and plants. We found that the GAU-α-helix correlation is also strong in non-human mammalian and vertebrate proteins but is much weaker or insignificant in S. cerevisiae and plants. The vertebrate correlations are of sufficient strength to enhance α-helix N-terminus prediction. Additional results, including the observation that the correlation is significantly enhanced when proteins that are known to be correctly expressed in recombinant procaryotic systems are excluded, suggest that the correlation is induced at the level of protein translation and folding and not at the nucleic acid level. To the best of our knowledge, it is not explicable by the canonical picture of protein expression and folding, suggesting the existence of a novel evolutionary selection mechanism. One possible explanation is that some α-helix N-terminal GAU codons may facilitate correct co-translational folding in vertebrates.     

枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响

目前,有关同义密码子使用偏性对蛋白质折叠的影响研究中,样本蛋白均来源于不同的物种。考虑到同义密码子使用偏性的物种差异性,选取枯草杆菌的核蛋白为研究对象。首先,将每条核蛋白按二级结构截取为α螺旋片段、β折叠片段和无规卷曲（α-β混合）片段,并计算其蛋白质折叠速率。然后,整理每个片段相应的核酸序列信息,计算其同义密码子使用度。在此基础上,分析枯草芽孢杆菌核蛋白的同义密码子使用偏性与蛋白质折叠速率的相关性。发现对于不同二级结构的肽链片段,都有部分密码子的使用偏性与其对应的肽链折叠速率显著相关。进一步分析发现,与肽链片段折叠速率显著相关的密码子绝大部分为枯草杆菌全序列或核蛋白序列的每一组同义密码子中使用度最高的密码子。结果表明,在蛋白质的折叠过程中,枯草芽孢杆菌的同义密码子使用偏性起着重要作用。     

Folding at the rhythm of the rare codon beat

The persistent difficulties in the production of protein at high levels in heterologous systems, as well as the inability to understand pathologies associated with protein aggregation, highlight our limited knowledge on the mechanisms of protein folding in vivo. Attempts to improve yield and quality of recombinant proteins are diverse, frequently involving optimization of the cell growth temperature, the use of synonymous codons and/or the co-expression of tRNAs, chaperones and folding catalysts among others. Although protein secondary structure can be determined largely by the amino acid sequence, protein folding within the cell is affected by a range of factors beyond amino acid sequence. The folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins and ligands, the ribosome, translocation through a pore membrane, redox conditions, among others. The translation rate as well as the translation machinery itself can dramatically affect protein folding, and thus the structure and function of the protein product. This review addresses current efforts to better understand how the use of synonymous codons in the mRNA and the availability of tRNAs can modulate translation kinetics, affecting the folding, the structure and the biological activity of proteins.     

Silent mutations affect in vivo protein folding in Escherichia coli

As an approach to investigate the molecular mechanism of in vivo protein folding and the role of translation kinetics on specific folding pathways, we made codon substitutions in the EgFABP1 (Echinococcus granulosus fatty acid binding protein1) gene that replaced five minor codons with their synonymous major ones. The altered region corresponds to a turn between two short alpha helices. One of the silent mutations of EgFABP1 markedly decreased the solubility of the protein when expressed in Escherichia coli. Expression of this protein also caused strong activation of a reporter gene designed to detect misfolded proteins, suggesting that the turn region seems to have special translation kinetic requirements that ensure proper folding of the protein. Our results highlight the importance of codon usage in the in vivo protein folding.     

Forbidden synonymous substitutions in coding regions

In the evolution of highly conserved genes, a few "synonymous" substitutions at third bases that would not alter the protein sequence are forbidden or very rare, presumably as a result of functional requirements of the gene or the messenger RNA. Another 10% or 20% of codons are significantly less variable by synonymous substitution than are the majority of codons. The changes that occur at the majority of third bases are subject to codon usage restrictions. These usage restrictions control sequence similarities between very distant genes. For example, 70% of third bases are identical in calmodulin genes of man and trypanosome. Third-base similarities of distant genes for conserved proteins are mathematically predicted, on the basis of the G+C composition of third bases. These observations indicate the need for reexamination of methods used to calculate synonymous substitutions.      

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

Genome variability and capsid structural constraints of hepatitis a virus

The number of synonymous mutations per synonymous site (K(s)), the number of nonsynonymous mutations per nonsynonymous site (K(a)), and the codon usage statistic (N(c)) were calculated for several hepatitis A virus (HAV) isolates. While K(s) was similar to those of poliovirus (PV) and foot-and-mouth disease virus (FMDV), K(a) was 1 order of magnitude lower. The N(c) parameter provides information on codon usage bias and decreases when bias increases. The N(c) value in HAV was about 38, while in PV and FMDV, it was about 53. The emergence of 22 rare codons in front of 8 in PV and 7 in FMDV was detected. Most of the conserved rare codons of the P1 region were strategically located at the carboxy borders of beta barrels and alpha helices, their potential function being the assurance of proper folding of the capsid proteins through a decrease in the translation speed. This strategic location was not observed for amino acids encoded by the conserved rare codons of the 3D region. The percentage of bases with low pairing number values was higher in the latter region, suggesting a role of the conserved rare codons in the maintenance of RNA structure. Many of the rare codons in HAV are among the most frequent in humans, unlike in PV or in FMDV. This fact may be explained by the lack of cellular shutoff in HAV. One hypothesis is that HAV has evolved in order to avoid competition with its host for cellular tRNAs.     

The imprint of codons on protein structure

The "central dogma" of biology outlines the unidirectional flow of interpretable data from genetic sequence to protein sequence. This has led to the idea that a protein's structure is dependent only on its amino acid sequence and not its genetic sequence. Recently, however, a more than transient link between the coding genetic sequence and the protein structure has become apparent. The two interact at the ribosome via the process of co-translational protein folding. Evidence for co-translational folding is growing rapidly, but the influence of codons on the protein structure attained is still highly contentious. It is theorised that the speed of codon translation modulates the time available for protein folding and hence the protein structure. Here, past and present research regarding synonymous codons and codon translation speed are reviewed within the context of protein structure attainment.     

同义密码子使用模式对蛋白产物表达及构象形成的影响*

鉴于遗传密码子的简并性能够将基因遗传信息的容量提升,同义密码子使用偏嗜性得以在生物体的基因组中广泛存在。虽然同义密码子之间碱基的变化并不能导致氨基酸种类的改变,在研究mRNA半衰期、编码多肽翻译效率及肽链空间构象正确折叠的准确性和翻译等这一系列过程中发现,同义密码子使用的偏嗜性在某种程度上通过精微调控翻译机制体现其遗传学功能。同义密码子指导tRNA在翻译过程中识别核糖体的速率变化是由氨基酸的特定顺序决定,并且在新生多肽链合成时,蛋白质共翻译转运机制同时调节其空间构象的正确折叠从而保证蛋白的正常生物学功能。某些同义密码子使用偏嗜性与特定蛋白结构的形成具有显著相关性,密码子使用偏嗜性一旦改变将可能导致新生多肽空间构象出现错误折叠。结合近些年来国内外在此领域的研究成果,阐述同义密码子使用偏嗜性如何发挥精微调控翻译的生物学功能与作用。     

Factors influencing synonymous codon and amino acid usage biases in Mimivirus

Synonymous codon and amino acid usage biases have been investigated in 903 Mimivirus protein-coding genes in order to understand the architecture and evolution of Mimivirus genome. As expected for an AT-rich genome, third codon positions of the synonymous codons of Mimivirus carry mostly A or T bases. It was found that codon usage bias in Mimivirus genes is dictated both by mutational pressure and translational selection. Evidences show that four factors such as mean molecular weight (MMW), hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in Mimivirus proteins. Based on our observation, we suggest that genes involved in translation, DNA repair, protein folding, etc., have been laterally transferred to Mimivirus a long ago from living organism and with time these genes acquire the codon usage pattern of other Mimivirus genes under selection pressure.     

Clonorchis sinensis: codon usage in nuclear genes

Codon usage in Clonorchis sinensis was analyzed using 12,515 codons from 38 coding sequences. Total GC content was 49.83%, and GC1, GC2 and GC3 contents were 56.32%, 43.15% and 50.00%, respectively. The effective number of codons converged at 51-53 codons. When plotted against total GC content or GC3, codon usage was distributed in relation to GC3 biases. Relative synonymous codon usage for each codon revealed a single major trend, which was highly correlated with GC content at the third position when codons began with A or U at the first two positions. In codons beginning with G or C base at the first two positions, the G or C base rarely occurred at the third position. These results suggest that codon usage is shaped by a bias towards G or C at the third base, and that this is affected by the first and second bases.     

Synonymous codon usage in different protein secondary structural classes of human genes: implication for increased non-randomness of GC3 rich genes towards protein stability

The relationship between the synonymous codon usage and different protein secondary structural classes were investigated using 401 Homo sapiens proteins extracted from Protein Data Bank (PDB). A simple Chi-square test was used to assess the significance of deviation of the observed and expected frequencies of 59 codons at the level of individual synonymous families in the four different protein secondary structural classes. It was observed that synonymous codon families show non-randomness in codon usage in four different secondary structural classes. However,when the genes were classified according to their GC3 levels there was an increase in non-randomness in high GC3 group of genes. The non-randomness in codon usage was further tested among the same protein secondary structures belonging to four different protein folding classes of high GC3 group of genes. The results show that in each of the protein secondary structural unit there exist some synonymous family that shows class specific codon-usage pattern. Moreover, there is an increased non-random behaviour of synonymous codons in sheet structure of all secondary structural classes in high GC3 group of genes. Biological implications of these results have been discussed.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

Transition states for folding of circular-permuted proteins

To explore the role of entropy and chain connectivity in protein folding, a particularly interesting scheme, namely, the circular permutation, has been used. Recently, experimental observations showed that there are large differences in the folding mechanisms between the wild-type proteins and their circular permutants. These differences are strongly related to the change in the intrachain connectivity. Some results obtained by molecular dynamics simulations also showed a good agreement with the experimental findings. Here, we use a topology-based free-energy functional method to study the role of the chain connectivity in folding by comparing features of transition states of the wild-type proteins with those of their circular permutants. We concentrate our study on 3 small globular proteins, namely, the alpha-spectrin SH3 domain (SH3), the chymotrypsin inhibitor 2 (CI2), and the ribosomal protein S6, and obtain exciting results that are consistent with the available experimental and simulation results. A heterogeneity of the interaction energies between contacts for protein CI2 and for protein S6 is also introduced, which characterizes the strong interactions between contacts with long loops, as speculated from experiments for protein S6. The comparison between the folding nucleus of the wild-type proteins and those of their circular permutants indicates that chain connectivity affects remarkably the shapes of the energy profiles and thus the folding mechanism. Further comparisons between our theoretical calculated phi(th) values and the experimental observed phi(exp) values for the 3 proteins and their permutants show that our results are in good agreement with experimental ones and that correlations between them are high. These indicate that the free-energy functional method really provides a way to analyze the folding behavior of the circular-permuted proteins and therefore the folding mechanism of the wild-type proteins.     

Single-Molecule Force Spectroscopy of Protein Folding

The use of force probes to induce unfolding and refolding of single molecules through the application of mechanical tension, known as single-molecule force spectroscopy (SMFS), has proven to be a powerful tool for studying the dynamics of protein folding. Here we provide an overview of what has been learned about protein folding using SMFS, from small, single-domain proteins to large, multi-domain proteins. We highlight the ability of SMFS to measure the energy landscapes underlying folding, to map complex pathways for native and non-native folding, to probe the mechanisms of chaperones that assist with native folding, to elucidate the effects of the ribosome on co-translational folding, and to monitor the folding of membrane proteins.     

Surprisingly high correlation between early and late stages in non-two-state protein folding

We collected quantitative kinetic data on early and late stages of folding in non-two-state proteins from the literature, and studied the relationship between the kinetics of the two stages. There was a surprisingly high correlation between the rate constants of these stages. The correlation coefficient of the logarithmic rate constants was as high as 0.97, which could not be caused by chance. We also studied relationships of the logarithmic rate constants of the two stages with native three-dimensional structures represented by the residue-residue contact map. There were again surprisingly high correlations between the logarithmic rate constants and the number of non-local contact clusters obtained from the contact maps. Because the number of non-local contact clusters represents overall arrangement of substructures in a native protein, the results strongly suggested the importance of the arrangement of the substructures for the kinetics of both early and late stages of protein folding.     

Genetic Code-guided Protein Synthesis and Folding in Escherichia coli

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10⁻⁸ m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.     

Synonymous codon usage in different protein secondary structural classes of human genes: Implication for increased non-randomness of GC<Subscript>3</Subscript> rich genes towards protein stability

The relationship between the synonymous codon usage and different protein secondary structural classes were investigated using 401 Homo sapiens proteins extracted from Protein Data Bank (PDB). A simple Chi-square test was used to assess the significance of deviation of the observed and expected frequencies of 59 codons at the level of individual synonymous families in the four different protein secondary structural classes. It was observed that synonymous codon families show non-randomness in codon usage in four different secondary structural classes. However, when the genes were classified according to their GC₃ levels there was an increase in non-randomness in high GC₃ group of genes. The non-randomness in codon usage was further tested among the same protein secondary structures belonging to four different protein folding classes of high GC₃ group of genes. The results show that in each of the protein secondary structural unit there exist some synonymous family that shows class specific codon-usage pattern. Moreover, there is an increased non-random behaviour of synonymous codons in sheet structure of all secondary structural classes in high GC₃ group of genes. Biological implications of these results have been discussed.