The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core

To investigate the relationships between sequence conservation, protein stability, and protein function, we have measured the thermodynamic stability, folding kinetics, and in vitro peptide-binding activity of a large number of single-site substitutions in the hydrophobic core of the Fyn SH3 domain. Comparison of these data to that derived from an analysis of a large alignment of SH3 domain sequences revealed a very good correlation between the distinct pattern of conservation observed at each core position and the thermodynamic stability of mutants. Conservation was also found to correlate well with the unfolding rates of mutants, but not to the folding rates, suggesting that evolution selects more strongly for optimal native state packing interactions than for maximal folding rates. Structural analysis suggests that residue-residue core packing interactions are very similar in all SH3 domains, which provides an explanation for the correlation between conservation and mutant stability effects studied in a single SH3 domain. We also demonstrate a correlation between stability and the in vivo activity of mutants, and between conservation and activity. However, the relationship between conservation and activity was very strong only for the three most conserved hydrophobic core positions. The weaker correlation between activity and conservation seen at the other seven core positions indicates that maintenance of protein stability is the dominant selective pressure at these positions. In general, the pattern of conservation at hydrophobic core positions appears to arise from conserved packing constraints, and can be effectively utilized to predict the destabilizing effects of amino acid substitutions.     

Sequence variations within protein families are linearly related to structural variations

It is commonly believed that similarities between the sequences of two proteins infer similarities between their structures. Sequence alignments reliably recognize pairs of protein of similar structures provided that the percentage sequence identity between their two sequences is sufficiently high. This distinction, however, is statistically less reliable when the percentage sequence identity is lower than 30% and little is known then about the detailed relationship between the two measures of similarity. Here, we investigate the inverse correlation between structural similarity and sequence similarity on 12 protein structure families. We define the structure similarity between two proteins as the cRMS distance between their structures. The sequence similarity for a pair of proteins is measured as the mean distance between the sequences in the subsets of sequence space compatible with their structures. We obtain an approximation of the sequence space compatible with a protein by designing a collection of protein sequences both stable and specific to the structure of that protein. Using these measures of sequence and structure similarities, we find that structural changes within a protein family are linearly related to changes in sequence similarity.     

基于Progressive多序列比对方法的求解多序列比对的启发式算法

在生物信息学研究中,生物序列比对问题占有重要的地位。多序列比对问题是一个NPC问题,由于时间和空间的限制不能够求出精确解。文中简要介绍了Feng和Doolittle提出的多序列比对算法的基本思想,并改进了该算法使之具有更好的比对精度。实验结果表明,新算法对解决一般的progressive多序列比对方法中遇到的局部最优问题有较好的效果。     

Evolutionary analysis of prokaryotic heat-shock transcription regulatory protein σ³²

Heat-stress to any living cell is known to trigger a universal defense response, called heat-shock response, with rapid induction of tens of different heat-shock proteins. Bacterial heat-shock genes are transcribed by the σ³²-bound RNA polymerase instead of the normal σ⁷⁰-bound RNA polymerase. In this study, the diversity in sequence, variation in secondary structure and function amongst the different functional regions of the proteobacterial σ³² family of proteins, and their phylogenetic relationships have been analyzed. Bacterial σ³² proteins can be subdivided into different functional regions which are referred to as regions 2, 3, and 4. There is a great deal of sequence conservation among the functional regions of proteobacterial σ³² family of proteins though some mutations are also present in these regions. Region 2 is the most conserved one, while region 4 has comparatively more variable sequences. In the present work, we tried to explore the effects of mutations in these regions. Our study suggests that the sequence diversities due to natural mutations in the different regions of proteobacterial σ³² family lead to different functions. So far, this study is the first bioinformatic approach towards the understanding of the mechanistic details of σ³² family of proteins using the protein sequence information only. This study therefore may help in elucidating the hitherto unknown molecular mechanism of the functionalities of σ³²family of proteins.     

On the role of structural information in remote homology detection and sequence alignment: new methods using hybrid sequence profiles

Structural alignments often reveal relationships between proteins that cannot be detected using sequence alignment alone. However, profile search methods based entirely on structural alignments alone have not been found to be effective in finding remote homologs. Here, we explore the role of structural information in remote homolog detection and sequence alignment. To this end, we develop a series of hybrid multidimensional alignment profiles that combine sequence, secondary and tertiary structure information into hybrid profiles. Sequence-based profiles are profiles whose position-specific scoring matrix is derived from sequence alignment alone; structure-based profiles are those derived from multiple structure alignments. We compare pure sequence-based profiles to pure structure-based profiles, as well as to hybrid profiles that use combined sequence-and-structure-based profiles, where sequence-based profiles are used in loop/motif regions and structural information is used in core structural regions. All of the hybrid methods offer significant improvement over simple profile-to-profile alignment. We demonstrate that both sequence-based and structure-based profiles contribute to remote homology detection and alignment accuracy, and that each contains some unique information. We discuss the implications of these results for further improvements in amino acid sequence and structural analysis.     

Recapitulation of protein family divergence using flexible backbone protein design

We use flexible backbone protein design to explore the sequence and structure neighborhoods of naturally occurring proteins. The method samples sequence and structure space in the vicinity of a known sequence and structure by alternately optimizing the sequence for a fixed protein backbone using rotamer based sequence search, and optimizing the backbone for a fixed amino acid sequence using atomic-resolution structure prediction. We find that such a flexible backbone design method better recapitulates protein family sequence variation than sequence optimization on fixed backbones or randomly perturbed backbone ensembles for ten diverse protein structures. For the SH3 domain, the backbone structure variation in the family is also better recapitulated than in randomly perturbed backbones. The potential application of this method as a model of protein family evolution is highlighted by a concerted transition to the amino acid sequence in the structural core of one SH3 domain starting from the backbone coordinates of an homologous structure.     

Computational and Experimental Evidence for the Evolution of a (βα)8-Barrel Protein from an Ancestral Quarter-Barrel Stabilised by Disulfide Bonds

The evolution of the prototypical (βα)₈-barrel protein imidazole glycerol phosphate synthase (HisF) was studied by complementary computational and experimental approaches. The 4-fold symmetry of HisF suggested that its constituting (βα)₂ quarter-barrels have a common evolutionary origin. This conclusion was supported by the computational reconstruction of the HisF sequence of the last common ancestor, which showed that its quarter-barrels were more similar to each other than are those of extant HisF proteins. A comprehensive sequence analysis identified HisF-N1 [corresponding to (βα)_1-2] as the slowest evolving quarter-barrel. This finding indicated that it is the closest relative of the common (βα)₂ predecessor, which must have been a stable and presumably tetrameric protein. In accordance with this prediction, a recombinantly produced HisF-N1 protein was properly folded and formed a tetramer being stabilised by disulfide bonds. The introduction of a disulfide bond in HisF-C1 [corresponding to (βα)_5-6] also resulted in the formation of a stable tetramer. The fusion of two identical HisF-N1 quarter-barrels yielded the stable dimeric half-barrel HisF-N1N1. Our findings suggest a two-step evolutionary pathway in which a HisF-N1-like predecessor was duplicated and fused twice to yield HisF. Most likely, the (βα)₂ quarter-barrel and (βα)₄ half-barrel intermediates on this pathway were stabilised by disulfide bonds that became dispensable upon consolidation of the (βα)₈-barrel.     

Consensus Protein Design without Phylogenetic Bias

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function.     

Clustal W—蛋白质与核酸序列分析软件

蛋白质与核酸的序列分析在现代生物学和生物信息学中发挥着重要作用,新的算法和软件层出不穷,本文介绍一个可运行在ＰＣ机上的完全免费的多序列比较软件－ＣｌｕｓｔａｌＷ,它不但可以进行蛋白质与核酸的多序列比较,分析不同序列之间的相似性关系,还可以绘制进化树。由于其灵活的输入输出格式、方便的参数设定和选择、详尽的在线帮助以及良好的可移植性,使得ＣｌｕｓｔａｌＷ在蛋白质与核酸的序列分析中得到了广泛应用。