功能性蛋白举例

自１９世纪中叶荷兰化学家ＧｅｒａｒｄｕｓＭｕｌ－ｄｅｒ从动物组织和植物体中提取出蛋白质以来,人们发现了越来越多的蛋白质,据估计生物界中蛋白质的种类可达１０１０～１０１２之多;在这如此众多的蛋白质中,功能性蛋白发挥着极其重要的生理功能。功能性蛋白也有人...     

氯霉素和四环素阻碍细菌蛋白分泌研究进展

氯霉素和四环素发挥活性的一个途径就是阻碍细菌蛋白质的分泌,其分泌功能是由其氨基端的信号序列决定的,该序列能将蛋白质引导到由SecY,E,G和A组成的转运蛋白复合体上。蛋白的转运还取决于融合蛋白的折叠特点,蛋白质转运到周质后的错误折叠可导致毒素聚集体形成,快速折叠还会使转运复合体发生拥堵,使所有的蛋白质分泌都受到抑制,导致细胞死亡。抗生素氯霉素和四环素处理细菌后会导致转运复合体中SecY的降解,造成致命的蛋白拥堵。现就抗生素氯霉素和四环素的干扰细菌蛋白质合成的作用机制以及导致SecY的降解来发挥阻碍细菌蛋白质分泌活性的一个新模式进行概述,以期为探讨新的靶向细菌的治疗方法提供科学依据。     

蛋白- 蛋白作用界面特征及界面预测研究进展

蛋白-蛋白界面与其余蛋白表面有明显的差别。本文对近年来国内外有关蛋白-蛋白界面几何学、物理学、化学、进化保守性等方面特征的研究概况及应用这些特征对单体中预测界面方法的研究进展于以综述。     

细胞膜蛋白与细胞骨架蛋白相互作用研究进展

细胞膜蛋白与胞浆骨架蛋白的相互作用对于维持细胞正常形态,细胞粘附与信号传导有重要作用,含有4.1/JEF结构域的蛋白4.1超家族与含有PDZ结构域的MAGUK蛋白家族能结合多种膜蛋白胞内区与胞浆蛋白,在膜蛋白与胞浆蛋白之间建立联系,对于细胞、细胞-细胞间连接的正常结构与功能的维持有着重要作用。     

SMC蛋白的结构和功能

近年来新发现的一类蛋白－染色体结构维持蛋白（SMC蛋白,structural maintenance of chromosome proteins)与染色体结构细胞周期性的动态变化紧密相关,它们参与有丝分裂染色体的集缩和分离,性染色体的剂量补偿效应,姐妹染色单体的内聚作用（cohesion),遗传重组和DNA修复等过程,本从生化特性和生物学功能两方面叙述了对SMC蛋白的研究。     

淀粉样前体蛋白和LRP6在大肠杆菌中的表达及其体外相互作用

 淀粉样前体蛋白 (APP)是阿尔茨海默氏病 (AD)发病过程中有重要作用的蛋白 .利用酵母双杂交的方法发现低密度脂蛋白受体相关蛋白 6(LRP6)羧基端可和 APP羧基端片段相互作用 .分别构建了 APP和 LRP6的原核表达载体 ,并利用大肠杆菌获得 GST- APP1 0 6、MBP- LRP6融合蛋白 .体外相互作用研究证实了 APP羧基端和 LRP6羧基端之间的结合 .这使与 AD相关的两个重要蛋白 apo E和 APP联系起来 ,并提示 LRP6可能在 APP代谢和 Aβ产生中起重要作用 .     

Structure–activity studies on the upstream splice junction of a semisynthetic intein

Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int^N fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (?1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N–S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (?1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.     

Equilibrium and kinetic studies of protein cooperativity using urea-induced folding/unfolding of a Ubq-UIM fusion protein

Understanding the origins of cooperativity in proteins remains an important topic in protein folding. This study describes experimental folding/unfolding equilibrium and kinetic studies of the engineered protein Ubq-UIM, consisting of ubiquitin (Ubq) fused to the sequence of the ubiquitin interacting motif (UIM) via a short linker. Urea-induced folding/unfolding profiles of Ubq-UIM were monitored by far-UV circular dichroism and fluorescence spectroscopies and compared to those of the isolated Ubq domain. It was found that the equilibrium data for Ubq-UIM is inconsistent with a two-state model. Analysis of the kinetics of folding shows similarity in the folding transition state ensemble between Ubq and Ubq-UIM, suggesting that formation of Ubq domain is independent of UIM. The major contribution to the stabilization of Ubq-UIM, relative to Ubq, was found to be in the rates of unfolding. Moreover, it was found that the kinetic m-values for Ubq-UIM unfolding, monitored by different probes (far-UV circular dichroism and fluorescence spectroscopies), are different; thereby, further supporting deviations from a two-state behavior. A thermodynamic linkage model that involves four states was found to be applicable to the urea-induced unfolding of Ubq-UIM, which is in agreement with the previous temperature-induced unfolding study. The applicability of the model was further supported by site-directed variants of Ubq-UIM that have altered stabilities of Ubq/UIM interface and/or stabilities of individual Ubq- and UIM-domains. All variants show increased cooperativity and one variant, E43N_Ubq-UIM, appears to behave very close to an equilibrium two-state.     

Tracing Primordial Protein Evolution through Structurally Guided Stepwise Segment Elongation

The understanding of how primordial proteins emerged has been a fundamental and longstanding issue in biology and biochemistry. For a better understanding of primordial protein evolution, we synthesized an artificial protein on the basis of an evolutionary hypothesis, segment-based elongation starting from an autonomously foldable short peptide. A 10-residue protein, chignolin, the smallest foldable polypeptide ever reported, was used as a structural support to facilitate higher structural organization and gain-of-function in the development of an artificial protein. Repetitive cycles of segment elongation and subsequent phage display selection successfully produced a 25-residue protein, termed AF.2A1, with nanomolar affinity against the Fc region of immunoglobulin G. AF.2A1 shows exquisite molecular recognition ability such that it can distinguish conformational differences of the same molecule. The structure determined by NMR measurements demonstrated that AF.2A1 forms a globular protein-like conformation with the chignolin-derived β-hairpin and a tryptophan-mediated hydrophobic core. Using sequence analysis and a mutation study, we discovered that the structural organization and gain-of-function emerged from the vicinity of the chignolin segment, revealing that the structural support served as the core in both structural and functional development. Here, we propose an evolutionary model for primordial proteins in which a foldable segment serves as the evolving core to facilitate structural and functional evolution. This study provides insights into primordial protein evolution and also presents a novel methodology for designing small sized proteins useful for industrial and pharmaceutical applications.     

Dual conformation of H2H3 domain of prion protein in mammalian cells

The concept of prion is applied to protein modules that share the ability to switch between at least two conformational states and transmit one of these through intermolecular interaction and change of conformation. Although much progress has been achieved through the understanding of prions from organisms such as Saccharomyces cerevisiae, Podospora anserina, or Aplysia californica, the criteria that qualify a protein module as a prion are still unclear. In addition, the functionality of known prion domains fails to provide clues to understand the first identified prion, the mammalian infectious prion protein, PrP. To address these issues, we generated mammalian cellular models of expression of the C-terminal two helices of PrP, H2 and H3, which have been hypothesized, among other models, to hold the replication and conversion properties of the infectious PrP. We found that the H2H3 domain is an independent folding unit that undergoes glycosylations and glycosylphosphatidylinositol anchoring similar to full-length PrP. Surprisingly, in some conditions the normally folded H2H3 was able to systematically go through a conversion process and generate insoluble proteinase K-resistant aggregates. This structural switch involves the assembly of amyloid structures that bind thioflavin S and oligomers that are reactive to A11 antibody, which specifically detects protein oligomers from neurological disorders. Overall, we show that H2H3 is a conformational switch in a cellular context and is thus suggested to be a candidate for the conversion domain of PrP.     

Single-molecule force spectroscopy reveals the individual mechanical unfolding pathways of a surface layer protein

Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra- and intermolecular mechanical stress. In gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopy-based single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.     

Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion

Ring-forming AAA(+) ATPases act in a plethora of cellular processes by remodeling macromolecules. The specificity of individual AAA(+) proteins is achieved by direct or adaptor-mediated association with substrates via distinct recognition domains. We investigated the molecular basis of substrate interaction for Vibrio cholerae ClpV, which disassembles tubular VipA/VipB complexes, an essential step of type VI protein secretion and bacterial virulence. We identified the ClpV recognition site within VipB, showed that productive ClpV-VipB interaction requires the oligomeric state of both proteins, solved the crystal structure of a ClpV N-domain-VipB peptide complex, and verified the interaction surface by mutant analysis. Our results show that the substrate is bound to a hydrophobic groove, which is formed by the addition of a single α-helix to the core N-domain. This helix is absent from homologous N-domains, explaining the unique substrate specificity of ClpV. A limited interaction surface between both proteins accounts for the dramatic increase in binding affinity upon ATP-driven ClpV hexamerization and VipA/VipB tubule assembly by coupling multiple weak interactions. This principle ensures ClpV selectivity toward the VipA/VipB macromolecular complex.     

The aggregation properties of Escherichia coli proteins associated with their cellular abundance

Proteins are key players in most cellular processes. Therefore, their abundances are thought to be tightly regulated at the gene-expression level. Recent studies indicate, however, that steady-state cellular-protein concentrations correlate better across species than the levels of the corresponding mRNAs; this supports the existence of selective forces to maintain precise cellular-protein concentrations and homeostasis, even if gene-expression levels diverge. One of these forces might be the avoidance of protein aggregation because, in the cell, the folding of proteins into functional conformations might be in competition with anomalous aggregation into non-functional and usually toxic structures in a concentration-dependent manner. The data in the present work provide support for this hypothesis because, in E. coli, the experimental solubility of proteins correlates better with the cellular abundance than with the gene-expression levels. We found that the divergence between protein and mRNAs levels is low for high-abundance proteins. This suggests that because abundant proteins are at higher risk of aggregation, cellular concentrations need to be stringently regulated by gene expression.     

Evolutionary remodeling of βγ-crystallins for domain stability at cost of Ca2+ binding

The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across βγ-crystallin members critical for their specialized functions.     

Protein kinases and protein phosphatases in prokaryotes: a genomic perspective

For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.