Functional Binding Surface of a β‐Hairpin VEGF Receptor Targeting Peptide Determined by NMR Spectroscopy in Living Cells

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast ¹⁵N‐edited NMR spectroscopic experiments. To this aim, ¹⁵N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope‐edited NMR spectroscopic experiments, including ¹⁵N relaxation measurements, allowed a precise characterization of the in‐cell HPLW epitope recognized by VEGFR2.     

Thermodynamic study of transthyretin association (wild‐type and senile forms) with heparan sulfate proteoglycan: pH effect and implication of the reactive histidine residue

The tetramer destabilization of transthyretin into monomers and its fibrillation are phenomena leading to amyloid deposition. Heparan sulfate proteoglycan (HSPG) has been found in all amyloid deposits. A chromatographic approach was developed to compare binding parameters between wild‐type transthyretin (wtTTR) and an amyloidogenic transthyretin (sTTR). Results showed a greater affinity of sTTR for HSPG at pH 7.4 compared with wtTTR owing to the monomeric form of sTTR. Analysis of the thermodynamic parameters showed that van der Waals interactions were involved at the complex interface for both transthyretin forms. For sTTR, results from the plot representing the number of protons exchanged vs pH showed that the binding mechanism was pH‐dependent with a critical value at a pH 6.5. This observation was due to the protonation of a histidine residue as an imidazolium cation, which was not accessible when TTR was in its tetrameric structure. At pH >6.5, dehydration at the binding interface and several contacts between nonpolar groups of sTTR and HSPG were also coupled to binding for an optimal hydrogen‐bond network. At pH <6.5, the protonation of the His residue from sTTR monomer when pH decreased broke the hydrogen‐bond network, leading to its destabilization and thus producing slight conformational changes in the sTTR monomer structure. Copyright © 2014 John Wiley & Sons, Ltd.     

与HBV X蛋白相互作用的细胞蛋白的筛选及其鉴定

参考GenBank核苷酸序列库中的HBV的核苷酸序列设计合成了一对对应于HBx基因的引物，从原发性肝癌(hepatocellular carcinoma，HCC)患者的血清中提取HBV基因组DNA作为模板，扩增HBx编码区并测定了核苷酸序列．将该编码区克隆到酵母双杂交系统的诱饵蛋白表达载体pGBKT7中，转入酵母细胞AH109，进行表型鉴定．然后通过Mating实验从已制备好用于酵母双杂交系统的肝cDNA表达文库中筛选与HBx相互作用的细胞蛋白，并用体外免疫共沉淀实验进一步验证．研究表明，分离得到的与HBx基因编码的蛋白相互作用的4种新的细胞蛋白，分别是醛缩酶B、C8α亚基、一种丝氨酸蛋白酶Hepsin和一种未知蛋白．     

Self-Interactions of Strands and Sheets

Physical strands or sheets that can be modelled as curves or surfaces embedded in three dimensions are ubiquitous in nature, and are of fundamental importance in mathematics, physics, biology, and engineering. Often the physical interpretation dictates that self-avoidance should be enforced in the continuum model, i.e., finite energy configurations should not self-intersect. Current continuum models with self-avoidance frequently employ pairwise repulsive potentials, which are of necessity singular. Moreover the potentials do not have an intrinsic length scale appropriate for modelling the finite thickness of the physical systems. Here we develop a framework for modelling self-avoiding strands and sheets which avoids singularities, and which provides a way to introduce a thickness length scale. In our approach pairwise interaction potentials are replaced by many-body potentials involving three or more points, and the radii of certain associated circles or spheres. Self-interaction energies based on these many-body potentials can be used to describe the statistical mechanics of self-interacting strands and sheets of finite thickness.     

用N-(3-P)NEM标记毛发角蛋白研究毛发中低硫蛋白和高硫蛋白的状态

本文用一种特异性标记蛋白质巯基的N-(3-P)NEM为荧光探针标记毛发角蛋白,经SDS-PAGE后,通过荧光薄层扫描确定N-(3-P)NEM与含巯基蛋白结合的部位。结果表明,毛发角蛋白中的低硫蛋白分子量在45,000～63,000范围,约占总蛋白量的49.6％～68.2％;高硫蛋白分子量在4,000～20,000范围,约占总蛋白量的18.9％～36.2％。除低硫和高硫蛋白以外,毛发角蛋白中还可能存在一种极低硫蛋白,分子量位于22,000～32,000范围。对人与5种动物的毛发角蛋白组分进行比较,证实人与动物的毛发角蛋白的主要差异在高硫蛋白,不同种属的动物毛发角蛋白中低硫蛋白和高硫蛋白的含量也不相同。作者认为毛发角蛋白中含巯基蛋白的差异可为动物分类学和法医学在进行毛发比较鉴别方面提供新的重要信息。     

Kinetics of proton transfer in a green fluorescent protein: A laser-induced pH jump study

The sub-millisecond protonation dynamics of the chromophore in S65T mutant form of the green fluorescent protein (GFP) was tracked after a rapid pH jump following laser-induced proton release from the caged photolabile compoundo-nitrobenzaldehyde. Following a jump in pH from 8 to 5 (which is achieved within 2 μs), the fluorescence of S65T GFP decreased as a single exponential with a time constant of ∼90 μs. This decay is interpreted as the conversion of the deprotonated fluorescent GFP chromophore to a protonated non-fluorescent species. The protonation kinetics showed dependence on the bulk viscosity of the solvent, and therefore implicates bulk solvent-controlled protein dynamics in the protonation process. The protonation is proposed to be a sequential process involving two steps: (a) proton transfer from solvent to the chromophore, and (b) internal structural rearrangements to stabilize a protonated chromophore. The possible implications of these observations to protein dynamics in general is discussed     

Intrinsic Fluorescence of Actin

In this work actin is used to illustrate connection of protein fluorescence characteristics with its structure. On one hand, it has been demonstrated what kind of information about the contribution of each tryptophan residues to the bulk fluorescence spectrum can be obtained from the special analysis of protein three-dimensional structure. On the other hand, potentials of intrinsic fluorescence for elucidation of proteins structure, dynamics and processes of folding-unfolding are shown. In particular, using this method a new essentially unfolded kinetic intermediate state of actin was detected and characterized, and the place of inactivated actin and its kinetic predecessor in the process of folding-unfolding was determined. It has been revealed that inactivated actin is not intermediate state between the native and completely unfolded states, as it has been accepted before, but a result of protein misfolding. On the basis of the obtained data a new model of actin folding-unfolding pathway has been proposed.     

酵母蛋白质网络的动力学性质

蛋白质-蛋白质、蛋白质-DNA相互作用网络决定了细胞中各种关键功能的执行．基于芽殖酵母(budding yeast saccharomyces cerevisiae)的蛋白质-蛋白质相互作用网络数据和相关的实验文献，我们建立了调控细胞周期和生命周期(cell cycle and life cycle)的蛋白质网络，并利用离散模型研究了该网络的动力学性质，研究表明：细胞周期网络的动力学性质具有很强的稳定性，约94％的蛋白质初态将演化到对应于生物学G1基态的稳定态，使其成为惟一的全局吸引点；同时，绝大多数的初态的演化路径都通过由G1激发态到G1基态的细胞周期演化路径，使细胞周期路径成为全局性的“吸引”路径。     

复制缺陷型人泡沫病毒载体辅助质粒pΔGP的构建及转染小肠癌细胞的研究

在人泡沫病毒原病毒全长克隆pHRSV13的基础上,缺失突变gag和pol基因,并且用SV40polyA加尾信号替代人泡沫病毒的3′LTR,构建辅助质粒pΔGP.将复制缺陷型人泡沫病毒载体质粒pGPSNI EGFP和辅助质粒pΔGP分别转染和共转染小肠癌HIC细胞系,荧光显微镜检测发现共转染pGPSNI EGFP和pΔGP的HIC细胞能够强烈表达绿色荧光蛋白,转染有复制缺陷型人泡沫病毒载体质粒pGPSNI EGFP的HIC细胞能够表达少量的绿色荧光蛋白,而转染有辅助载体pΔGP的HIC细胞不表达绿色荧光.结果证明复制缺陷型人泡沫病毒载体的构建成功,表明人泡沫病毒env基因3′端的内部启动子IP具有弱启动子的活性,并且bel基因产生的调控蛋白能够反式激活人泡沫病毒内部启动子IP和5′LTR的启动子.