Small angle neutron scattering studies on protein denaturation induced by different methods

Small angle neutron scattering (SANS) has been used to study conformational changes in protein bovine serum albumin (BSA) as induced by varying temperature and in the presence of protein denaturating agents urea and surfactant. BSA has prolate ellipsoidal shape and is found to be stable up to 60°C above which it denaturates and subsequently leads to aggregation. The protein solution exhibits a fractal structure at temperatures above 64°C, with fractal dimension increasing with temperature. BSA protein is found to unfold in the presence of urea at concentrations greater than 4 M and acquires a random coil Gaussian chain conformation. The conformation of the unfolded protein in the presence of surfactant has been determined directly using contrast variation SANS measurements by contrast matching surfactant molecules. The protein acquires a random coil Gaussian conformation on unfolding with its radius of gyration increasing with increase in surfactant concentration      

Powder diffraction from a continuous microjet of submicrometer protein crystals

Atomic‐resolution structures from small proteins have recently been determined from high‐quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40 , 133–143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129 , 11865–11871]. While powder diffraction data have been obtained from batch samples of small crystal‐suspensions, which are exposed to X‐rays for long periods of time and undergo significant radiation damage, the proof‐of‐concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow‐focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X‐ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest‐order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free‐electron X‐ray laser schemes, and for serial crystallography using a single‐file beam of aligned hydrated molecules.     

Coherent X‐ray diffractive imaging of protein crystals

The technique of coherent X‐ray diffraction imaging (CXDI) has recently shown great promise for the study of inorganic nanocrystals. In this work the CXDI method has been applied to the study of micrometer‐size protein crystals. Finely sampled diffraction patterns of single crystals were measured and iterative phase‐retrieval algorithms were used to reconstruct the two‐dimensional shape of the crystal. The density maps have limited reproducibility because of radiation damage, but show clear evidence for crystal facets. Qualitative analysis of a number of single‐crystal diffraction peaks indicates the presence of inward surface contraction on 2 µm size crystals. A survey of several hundred diffraction patterns yielded a number of examples with dramatic single‐sided streaks, for which a plausible model is constructed.     

蛋白质支链动力学快运动的核磁共振研究

蛋白质的功能不仅取决于其结构,而且受到其构像及其变化的影响. 许多生物化学过程就是由于蛋白质结构的一些动力学变化而完成,如蛋白质-蛋白质,蛋白质-药物配体之间的相互作用. 因此分析蛋白质的动力学变化,就能够对其参与的生化过程进行分析. 作为动力学研究的有力工具之一,核磁共振能够分辨到原子范围内的从千秒到皮秒时间范围的运动过程,因此在动力学研究中有着不可替代的作用. 本文仅就核磁共振在蛋白质支链快运动方法（ps-ns）研究方面的进展进行总结,以期阐明核磁共振的在支链动力学研究中的发展现状.     

基于高分辨质谱技术的婴幼儿食品中过敏原蛋白质的高灵敏检测

基于高效液相色谱-串联质谱(LC-MS/MS)技术,选择稳定性好、灵敏度高的特征肽段,利用平行反应监测(PRM)技术,实现了多类过敏原蛋白质的高灵敏度同时检测,并成功应用于婴幼儿食品中过敏原成分的分析。对于婴幼儿食品中蛋白质的提取,与传统的丙酮沉淀法比,采用膜上原位样品预处理方法(i-FASP)可实现更高的蛋白质提取效率和抗干扰能力。所检测的过敏原蛋白质的定量限(LOQ)最小可达到0.028 mg/L,其线性范围最宽可跨越4个数量级,且线性关系良好(相关系数R~2≥0.99)。该方法为食品中过敏原蛋白质组学快速分析提供了一种可靠的分析方法。     

聚乳酸-聚半胱氨酸混纺纤维毡用于谷胱甘肽S-转移酶的捕获和洗脱

本文研究一种分离纯化GST（谷胱甘肽S-转移酶）的方法：将嵌段聚合物聚左旋乳酸-聚半胱氨酸（PLLA—PCys）电纺丝制成超细纤维,将还原型谷胱甘肽（GSH）通过二硫键偶联到纤维上,再将纤维填充玻璃管,制成吸附柱;含有GST的混合溶液通过吸附柱时,GST被纤维捕获;再用含GSH的溶液流过吸附柱,GST被洗脱下来,经冷冻干燥获得GST纯品．用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳进行检测．结果表明,键合了GSH的PLLA—PCys混纺纤维毡能够从蛋白混合溶液中专一性地捕获GST,被捕获的GST可以有效地回收．所以,此种键合了GSH的电纺丝纤维可望用于GST或以GST为标签的蛋白的分离、纯化以及检测．     

A proteome-wide analysis of domain architectures of prokaryotic single-spanning transmembrane proteins

We performed a proteome-wide survey of the domain architectures in single-spanning transmembrane (TM) proteins (single-spannings) from 87 sequenced prokaryotic (Bacterial and Archaean) genomes by assigning Pfam domains to their N-tail and C-tail loops. Out of 14,625 single-spannings, 3,516 sequences have at least one domain assigned, and no domains were assigned to 7,850, with the remaining 3,259 with less reliable assignment. In the domain-assigned sequences, 3116 sequences are with at most two domains, and the other 400 sequences with more than two. The assigned domains distribute over 651 Pfam families, which account for 11.4% of the total Pfam-A families. Among the 651 families are mostly soluble-protein-originated ones, but only 21 families are unique to TM proteins. The occurrence frequency of the individual domain families follows a power-law, that is, 264 families occur only once, 106 just twice, and the families appeared more than 30 times are counted by only 39. It is found that the great majority of the sequences having one or two domains are of the type II topology with the C-tail loop containing domains on it. On the contrary, the N-tail loop of the same type topology seldom carries domains. Importantly, the assigned domains are always found on the tail loops longer than 60 residues, even for the small domains with less than 30 residues. There are still as many as 5,800 sequences without assigned domains in spite of having at least one long tail, on which no less than 1,000 novel domain families are expected most likely to lie concealed unknown yet. We also investigated the domain arrangement preference and the domain family combination patterns in 'singlets' (single-spannings with one assigned domain) and 'doublets' (with two domains).     

α螺旋蛋白质螺旋链模型的精确解组

文章运用Maple语言程序,在没有假设的条件下,得到了α螺旋蛋白质螺旋链运动模型方程组的行波精确解组,它涵盖了所有的耦合解组与非耦合解组,具有任意性.耦合解组的算例函数及其特性分析,解释了α螺旋蛋白质螺旋链运动模型的行波孤立子解的耦合效应,揭示了增加、稳定和控制蛋白质活性和功能的方向,文章的研究方法,为求解生物大分子螺旋链运动模型的行波精确解组探索了溪径.     

人工金属酶分子设计新进展:肌红蛋白研究实例分析

金属酶在生物体中发挥着多种至关重要的作用,而人工金属酶的分子设计能够调控和拓展天然金属酶的功能,甚至创造出功能更为优越的新型酶分子。肌红蛋白（Mb）是作为血红素蛋白或其他金属蛋白分子设计的理想蛋白模型。近些年,基于Mb蛋白骨架的人工金属酶的分子设计逐渐发展了多种研究策略,包括设计氢键网络、金属结合位点、分子内二硫键、利用蛋白翻译后修饰、引入非天然氨基酸和非天然辅基等。本文着重综述这些方面的最新研究进展,可以帮助我们深刻认识金属酶的结构与功能关系,同时掌握人工金属酶分子设计的思路与方法,从而有助于推动这一领域的快速发展。     

The characteristics of molten globule states and folding pathways strongly depend on the sequence of a protein

ABSTRACT

The majority of proteins perform their cellular function after folding into a specific and stable native structure. Additionally, for many proteins less compact ‘molten globule’ states have been observed. Current experimental observations show that the molten globule state can show varying degrees of compactness and solvent accessibility; the underlying molecular cause for this variation is not well understood. While the specificity of protein folding can be studied using protein lattice models, current design procedures for these models tend to generate sequences without molten globule-like behaviour. Here we alter the design process so the distance between the molten globule ensemble and the native structure can be steered; this allows us to design protein sequences with a wide range of folding pathways, and sequences with well-defined heat-induced molten globules. Simulating these sequences we find that (1) molten globule states are compact, but have less specific configurations compared to the folded state, (2) the nature of the molten globule state is highly sequence dependent, (3) both two-state and multi-state folding proteins may show heat-induced molten globule states, as observed in heat capacity curves. The varying nature of the molten globules and typical heat capacity curves associated with the transitions closely resemble experimental observations.