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.     

Sequence optimization for native state stability determines the evolution and folding kinetics of a small protein

Investigating the relative importance of protein stability, function, and folding kinetics in driving protein evolution has long been hindered by the fact that we can only compare modern natural proteins, the products of the very process we seek to understand, to each other, with no external references or baselines. Through a large-scale all-atom simulation of protein evolution, we have created a large diverse alignment of SH3 domain sequences which have been selected only for native state stability, with no other influencing factors. Although the average pairwise identity between computationally evolved and natural sequences is only 17%, the residue frequency distributions of the computationally evolved sequences are similar to natural SH3 sequences at 86% of the positions in the domain, suggesting that optimization for the native state structure has dominated the evolution of natural SH3 domains. Additionally, the positions which play a consistent role in the transition state of three well-characterized SH3 domains (by phi-value analysis) are structurally optimized for the native state, and vice versa. Indeed, we see a specific and significant correlation between sequence optimization for native state stability and conservation of transition state structure.     

金黄色葡萄球菌噬菌体vB_Sau_P68的基因组分析和裂解酶

【背景】金黄色葡萄球菌是常见的人畜共患条件致病菌,随着多耐药菌株分离率的增长,研发与抗生素作用模式不同的抗菌剂迫在眉睫。【目的】分离高效且特异性强的金黄色葡萄球菌噬菌体,对其进行功能注释,并对其编码的裂解酶进行功能验证。【方法】通过对噬菌体全基因组序列进行分析找到裂解酶基因,利用原核表达系统对其编码的2个裂解酶蛋白进行克隆,用SDS-PAGE与蛋白免疫印迹法(Western blotting)鉴定目的蛋白是否表达,并采用单斑法验证其裂解活性。【结果】本研究的噬菌体为一株新的金黄色葡萄球菌噬菌体,命名为vB＿Sau＿P68,该基因组全长为139 409 bp,GC含量为31.0%,编码220个开放阅读框(open reading frame,ORF),透射电镜观察具有正二十面体头部和收缩性尾部,形态学分类属于肌尾噬菌体。该噬菌体编码2个裂解酶基因,分别具有CHAP催化结构域与SH3＿5结合结构域,SDS-PAGE与Western blotting表明Lys161能够表达且有裂解活性,Lys163则无法外源表达。对Lys161序列进行分析,该裂解酶无信号肽,无跨膜区域,以无规则卷曲为主。【...     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase

The intron/exon organization of the human gene for glycogen phosphorylase has been determined. The segments of the polypeptide chain that corresponds to the 19 exons of the gene are examined for relationships between the three-dimensional structure to the protein and gene structure. Only weak correlations are observed between domains of phosphorylase and exons. The nucleotide binding domains that are found in phosphorylase and other glycolytic enzymes are examined for relationships between exons of the genes and structures of the domains. When mapped to the three-dimensional structures, the intron/exon boundaries are shown to be widely distributed in this family of protein domains.     

The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 Å for all backbone atoms and 0.40 Å for the backbone atoms of the central -sheet. The core structure of the SH2 domain contains an antiparallel -sheet flanked by two parallel -helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.