尿素诱导的蛋白质变性现象的研究

通过对典型蛋白质变性实验结果的分析，发现在蛋白质变性研究领域中普遍使用的二态模型所描述的过程，与真实蛋白质变性过程并不相符．在分析已有的实验结果的基础上，给出了反映蛋白质变性过程特征的新表示形式．这一形式表明蛋白质分子变性是一个构象渐变的过程，同时强调在一定浓度的尿素溶液中蛋白质分子均处于相同的构象态．从尿素分子与蛋白质肽链上的相邻二肽形成双氢键的假设出发，利用吸附反应动力学方法，得到了描述蛋白质变性特征的物理量与尿素浓度关系的表示式．结果表明理论值与实验值符合较好，说明建立的模型可较好地描述尿素导致的蛋白质变性现象．     

蛋白质热变性现象的研究

从构象渐变的观点,对蛋白质热变性现象进行了系统的分析,发现以前用二态模型无法解释的实验数据,可以用渐变的观点做出合理的解释.给出在实验中可以进一步验证热变性过程具有构象渐变特征的预测结果.     

蛋白质变性的渐变模型

通过对典型蛋白质变性实验的分析 ,证明了在目前蛋白质变性研究领域中普遍使用的二态模型与实际变性过程不符 .在分析已有实验结果的基础上 ,给出了反映蛋白质变性过程特征的新模型———渐变模型 ,该模型显示出蛋白质分子变性是一个构象渐变的连续稳态过程 .     

伸展态β-乳球蛋白重折叠过程的动力学特性

从蛋白质变性的渐变模型及构象态渐变的观点出发 ,对伸展态 β -乳球蛋白的重折叠过程进行了分析 ,发现蛋白质分子从伸展态到最终折叠态历经一系列中间稳定态 .尿素浓度梯度电泳实验结果显示 ,存在两条不同的折叠途径 .在不同的尿素浓度范围内 ,蛋白质分子的折叠过程具有不同的动力学特性     

用荧光法研究各种因素对赤豆α-球蛋白构象的影响

研究蛋白质变性,虽然已有五十多年的历史,但至今仍然是一个重要的研究课题。过去,对蛋白质变性的研究,多偏重于动物及微生物的蛋白,对植物蛋白的变性研究不多。用荧光法研究一些变性因素对赤豆α-球蛋白分子构象的影响,讫今未见报道。本文报道了这方面的研究结果。     

蛋白质结构域划分方法及在线服务综述

蛋白质结构域是研究蛋白质结构、功能与进化的基本单位,不同的结构域可组合出更为复杂的蛋白质分子.划分蛋白质结构域后,可以从结构域的角度研究蛋白质的结构、功能与进化,降低了研究复杂度.根据已知结构的蛋白质统计,有约40%的为多结构域蛋白质,其中还存在一级结构上不临近的氨基酸序列出现在同一个结构域的情况,即不连续结构域.文章给出了当前国内外有关蛋白质结构域边界预测、不连续结构域检测及结构域数据库与在线服务的研究进展,供相关研究者参考.     

α-淀粉酶及α-糜蛋白酶的脲变动力学研究

用疏水色谱（HIC）对8.0mol/L脲变性的α－淀粉酶（α-Amy)和α－糜蛋白酶（α－Chy)的变性动力学进行了研究。结果表明：α－Amy的变性为平缓的渐变过程，其变体数目较多，且绝大多数变体的疏水性都小于天然蛋白；α－Chy是骤变（可能是瞬间变性）过程，相对而言α－Chy的变体数目较少，且疏水性均大于天然蛋白。认为两种蛋白不同的脲变动力学行为，可能是因蛋白中α－螺旋含量以及蛋白质自身稳定性不同所致。     

脲对含内蛋白子前体蛋白剪切的影响

研究了含内蛋白子的前体蛋白［麦芽糖结合蛋白－内蛋白子－几丁质酶结合区（ＭＹＢ）］在脲溶液中的分子构象及变性和复性过程中剪切活性与光谱变化的关系。结果表明，剪切活性随脲浓度的增加而逐渐丧失；当脲浓度大于６ｍｏｌ／Ｌ时，活性完全丧失。变性过程中Ａ２８０ｎｍ随变性程度增加而增强，复性后Ａ２８０ｎｍ与天然态Ａ２８０ｎｍ值相近，剪切活性随之恢复。说明构象松散先于剪切活性，可能在重折叠时，构象核化对剪切活性致关重要。     

明胶蛋白变性的红外光谱解析及应用

通过红外光谱法研究了明胶蛋白多肽链中肽键的三维空间构象维系及氢键所起的重要作用；但这种构象并不稳定，在使用肉桂酰氯对亚胺基进行酰基化并光照交联成体型结构以后，明胶蛋白出现变性而呈非水溶性．这种交联变性在红外光谱上得到证实，并为明胶在制版工艺应用上提出一种新的研究方法．     

分子伴侣的研究进展

文章综述了分子伴侣特别是HSP70分子伴侣系统的结构、功能、作用机理及应用方面的研究进展。分子伴侣能结合和稳定另一种蛋白质的不稳定构象，促进新生多肽链的正确折叠，因而在辅助蛋白质复性以及免疫保护等方面有很重要的作用。HSP70分子伴侣能够帮助细胞内新生蛋白的折叠和跨膜运输、蛋白质多聚体结构的装配和解装配，并能在胁迫下维持蛋白质的特殊构象，防止未折叠的蛋白质变性和使聚集的蛋白质溶解复性。     

从高分子构象与动力学到蛋白质折叠

针对作用在聚合物刷上的键拉力研究表明作用在接枝基面上的力随着聚合物刷接枝密度的增大反而减小,然而尾端单体上的拉伸张力并没有消失.高分子的构象和动力学转变决定了其物性和多种多样的应用,而生物大分子蛋白质作为由二十种不同属性的氨基酸构成的序列,更是具有由其序列所决定的特别的三维自然结构.本文就聚合物刷、聚合物纳米复合材料、聚合物网络等几种高分子体系的构象与动力学过程,及蛋白质构象和其折叠与去折叠的动力学过程做了介绍.特别是蛋白质的折叠与去折叠速率在单分子操纵实验中受到拉力的调控,通过测量这种拉力依赖的动力学过程、蛋白质的自由能曲面和折叠去折叠路径可以得到系统全面的研究.本文以肌肉蛋白titin的免疫球蛋白结构域I27为例对蛋白质折叠研究进行了阐述.     

An improved method for measuring the stability of a three-state unfolding protein

In the current three-state protein unfolding model, the two transitions are considered to be independent and each transition is fitted to a two-state unfolding model. This three-state unfolding process is therefore composed of two sequential two-state unfolding processes. In this paper, a modified method is presented to determine the value of the unfolding free energy [δG _total⁰(H₂O)] for the three-state unfolding equilibrium of proteins. This method is demonstrated on the apoCopC protein mutant, Y79W-W83F-Cu, which unfolds via a three-state process. The value of ΔG _total⁰(H₂O) calculated using the modified method was found to be more accurate in determining ΔG _total⁰(H₂O) than the previously reported method.     

Kinetic redistribution of native and misfolded RNAs by a DEAD-box chaperone

DExD/H-box proteins are ubiquitously involved in RNA-mediated processes and use ATP to accelerate conformational changes in RNA. However, their mechanisms of action, and what determines which RNA species are targeted, are not well understood. Here we show that the DExD/H-box protein CYT-19, a general RNA chaperone, mediates ATP-dependent unfolding of both the native conformation and a long-lived misfolded conformation of a group I catalytic RNA with efficiencies that depend on the stabilities of the RNA species but not on specific structural features. CYT-19 then allows the RNA to refold, changing the distribution from equilibrium to kinetic control. Because misfolding is favoured kinetically, conditions that allow unfolding of the native RNA yield large increases in the population of misfolded species. Our results suggest that DExD/H-box proteins act with sufficient breadth and efficiency to allow structured RNAs to populate a wider range of conformations than would be present at equilibrium. Thus, RNAs may face selective pressure to stabilize their active conformations relative to inactive ones to avoid significant redistribution by DExD/H-box proteins. Conversely, RNAs whose functions depend on forming multiple conformations may rely on DExD/H-box proteins to increase the populations of less stable conformations, thereby increasing their overall efficiencies.     

Energetic landscape of alpha-lytic protease optimizes longevity through kinetic stability.

During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial alpha-lytic protease (alphaLP) these two processes have become decoupled. The native state of alphaLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 approximately 1,800 years) to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving alphaLP trapped in its native state by a large kinetic unfolding barrier (t1/2 approximately 1.2 years). alphaLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of alphaLP by the optimal suppression of proteolytic sensitivity.     

盐生杜氏藻和衣藻双结构域NAD+-GPD基因的生物信息学分析

盐生杜氏藻（Dunaliella salina）是极端耐盐的单细胞真核绿藻,依赖于NAD的3－磷酸甘油脱氢酶（NAD+-GPD）是盐生杜氏藻调渗物质——甘油合成的关键酶.盐生杜氏藻NAD+-GPD基因是第一个被发现含双结构域（SerB和GPD）的GPD基因.而双结构域可能是盐生杜氏藻具有极强耐盐性和快速合成甘油的关键.通过与莱茵衣藻基因组数据库比对,发现莱茵衣藻（Chlamydomonas reinhardtii）中也存在具有SerB和GPD结构域的NAD+-GPD基因序列.在对两个物种NAD+-GPD基因的基因结构、mRNA组成成分、编码蛋白及其理化性质和编码蛋白结构的分析中,发现二者具有较高的相似性.针对目前仅在盐藻和衣藻中发现含SerB和GPD结构域的NAD+-GPD基因这一现象,分别对SerB和GPD结构域同源基因的系统进化进行了分析.     

Enthalpy-entropy compensation in protein unfolding

Enthalpy-entropy compensation was found to be a universal law in protein unfolding based on over 3000 experimental data. Water molecular reorganization accompanying the protein unfolding was suggested as the origin of the enthalpy-entropy compensation in protein unfolding. It is indicated that the enthalpy-entropy compensation constitutes the physical foundation that satisfies the biological need of the small free energy changes in protein unfolding, without the sacrifice of the bio-diversity of proteins. The enthalpy-entropy compensation theory proposed herein also provides valuable insights into the Privalov’s puzzle of enthalpy and entropy convergence in protein unfolding.     

Nir2碳端结构域的晶体结构

Nir2蛋白具有多个结构域，在生物体中可能发挥多种生理功能。本实验用MAD软件对大肠杆菌BL21（plys）原核表达的Nir2蛋白碳端进行了晶体结构分析。结果显示，硒蛋氨酸取代蛋白晶体及未取代蛋白晶体同属空间群P212121,晶胞参数a=109.468 Å, b=120.621 Å, c=70.315 Å，分辨率分别为2.2 Å和2.7 Å。一个不对称单位含有三个分子，含水量为48%。Nir2蛋白碳端由C片层和N片层构成，其中C片层与钙ATPase的催化区域P相似，N片层为许多不相关的功能性蛋白所共有。研究证明二者之间的静电作用是影响Nir2蛋白碳端与Pyk2 FERM区域结合的主要因素。该晶体结构的分析结果为进一步研究Nir2蛋白碳端的潜在功能提供了结构基础。     

Protein folding and misfolding

The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.     

Mechanical unfolding intermediates in titin modules

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by approximately 7 A before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate' Steered molecular dynamics simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 A, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by approximately 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.     

Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.