HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.     

别构酶ATCase与底物分子协同结合过程的构象变化特性

利用可描述协同结合特性的动力学方法,得到了反映单个别构酶ATCase分子的结合分数与底物浓度之间的关系式.表明在确定的底物浓度条件下,单个别构酶ATCase分子处于确定的构象态,随着底物浓度的增加,别构酶ATCase分子将经历一系列中间态达到饱和构象态,S型结合分数曲线实际上显示了单个别构酶ATCase分子与底物分子的协同结合特性.利用所得模型分析了调节物分子对底物协同结合过程的影响,合理解释了结合分数曲线随调节物浓度变化而发生改变的实验结果,与别构酶ATCase分子具有的结构及所产生的生物学功能是自给的.     

Making antibody fragments using phage display libraries

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.     

与反应机理有关的计量学和热力学几个关系的论证

本文:(1)独立地用线性代数中的几何方法.较容易地证明了动力学中的一个新计量关系,k=ρ+π+ι=n+v。(2)对复杂反应的热力学平衡常数和机理中基元反应的动力学平衡常数之间的联系,提出一个新关系式。     

Cu2+对香豆素与BSA相互作用的影响

采用荧光光谱法研究了Cu^2＋对香豆素类中药有效成分的母核化合物香豆素与牛血清白蛋白（BSA）相互作用的影响．实验结果表明，有Cu^2＋存在时，香豆素对BSA内源荧光的猝灭过程是以静态猝灭为主，香豆素与BSA结合过程的表观结合常数KA数值为lO^4量级，结合位点数n的范围为2．9～3．4，香豆素-BSA的分子间相互作用力类型仍是以疏水作用为主，熵增加是该作用过程的主要热力学驱动因素；与不合Cu＾２＋的相比，香豆素对BSA内源荧光的静态猝灭常数KP、表观结合常数KA都明显增大，但结合位点数和分子间作用力类型没有明显改变，表明Cu＾2＋可能参与了香豆素与BSA的结合过程．     

气体普朗特数变化规律的初步研究

反映流体中能量和动量迁移过程相互影响的无因次组合数－普朗特数,在热力计算中具有重要的作用,作者从分子运动理论角度出发,对普朗特数进行了分析,通过氩,氮和水蒸气３种常见物质的普朗特数的变化情况,对普朗特数的一些基本变化规律进行了初步研究,并提出了适用于这３种物质的普朗特数变化规律的经验公式。     

鱼腥草中酮类活性成分与牛血清蛋白相互作用特征

运用荧光光谱和紫外光谱,分别在血清蛋白的等电点以下(pH3.78)和以上(pH7.40),研究了鱼腥草的活性成分甲基正壬酮(2UC)和十二酮(2DC)与牛血清蛋白(BSA)的相互作用.结果显示在等电点前后,(2UC)和(2DC)与BSA的结合呈现可饱和模式,但对荧光的猝灭模式不同,而且结合的饱和浓度也不同.285 K和pH7.40下,二者在浓度较低时呈现明显的正协同作用,而在301 K时没有这种现象.计算了不同温度下结合的热力学参数△H0,△G0,△S0,结果显示疏水作用是主要的作用力.猝灭常数显示出酸度、温度和烃链长度的依赖性.考察了常见金属离子和中药活性成分对结合的影响.结果显示当共存锌离子、ANS和甘草次酸时,两种成分的加入会引起最大荧光发射波长的强烈兰移.试验表明脂肪酸2结合位(fatty site 2)是它们的一个结合位点,而药物结合位点Ⅱ(siteⅡ)可能是它们的另一结合位点.     

综合化学实验设计:单/双核铜配合物的温度控制合成、表征及转化

化学类专业本科生在理论课学习中，对反应的热力学产物和动力学产物有了一定的理论认识，但目前鲜有实验能够使其直观体会2种产物在制备时的差异．本实验以氯化铜和邻氨基苯甲酰胺为原料，水和乙醇为溶剂，通过仅改变反应温度，分别在60 ℃水浴加热和冰水浴冷却条件下得到了红色的热力学产物二(μ-氯)·二[氯·(邻氨基苯甲酰胺)合铜(Ⅱ)] ( 1 )和黄绿色的动力学产物二氯·二(邻氨基苯甲酰胺)合铜(Ⅱ) ( 2 )，产率分别为80.4%和76.4%．采用络合滴定法和仪器分析方法对产物进行了表征测试，结果表明在不同温度下可制备2种不同组成、结构和性质的热力学和动力学产物，且在加热条件下，配合物 2 可以转化为配合物 1 ．本设计为化学类相关专业本科生综合实验教学提供了一个成功案例，可加深学生对结构与性能的科学关联以及构效关系的认识和理解．      

胰蛋白酶水解牛血清白蛋白过程集总动力学研究

在间歇搅拌反应器中用胰蛋白酶水解牛血清白蛋白 ,考察了反应温度、底物浓度与酶用量等操作参数对水解度的影响规律。在此基础上 ,根据水解产物的相对分子质量分布将其归入四个集总组分 ,建立了此反应体系的四集总动力学模型。按照自下而上分层估算的原则 ,采用Marquardt法估算出反应网络中各反应的动力学常数。结果表明 ,产物空间体积的大小是影响反应速率的主要因素之一 ,动力学常数与温度的关系能较好地符合阿累尼乌斯关系式。经不同原料组成和反应条件的实验验证 ,各集总组分浓度的模型计算值与实验值较为吻合 ,这说明所建立的集总动力学模型能较为有效地预测反应网络的产物分布     

跨临界CO2引射制冷循环临界背压分析

为了优化设计或控制引射系统的性能,建立了跨临界CO2引射制冷循环中引射器性能的设定背压模型,定义引射器临界背压为忽略引射器出口动能时的引射器迭代背压,分析了引射器背压和出口速度对系统性能的影响.结果表明:当引射器背压低于临界值时,引射系数保持不变,且在不同系统高压侧压力下均成立;系统性能系数(COP)随着背压升高而增大,在系统状态可控的情况下引射器背压应尽量靠近临界值.同时,不同背压下COP随高压侧压力变化的趋势相同,背压作为设计或控制参数不会影响高压侧压力的优化.引射器出口动能会因流动摩擦和冲击而转化成热量并造成系统的热力学损失,背压选取不当会造成较大的引射器出口速度,且随着背压降低,出口速度增大,随着系统高压侧压力升高,出口速度先急剧降低,再缓慢变化.     

噬菌体展示蛋白质芯片的无标记检测

为获得丰富的特异识别分子和实现无标记、高灵敏度检测,利用噬菌体展示技术,选择在pⅢ蛋白上展示12肽的噬菌体M13作探针,采用羧基-氨基共价耦联法制备噬菌体展示蛋白质芯片.在此基础上,与表面等离子体共振传感技术结合,使用Biacore3000仪器,检测噬菌体展示蛋白质芯片与特异性抗体的反应,测定反应动力学常数.实验结果表明:当抗体浓度为0.4 μmol·L-1时,响应信号可达365±8个单位;不同浓度的抗体与芯片反应的数据较好地与S型曲线吻合.该方法可望用于噬菌体展示蛋白质芯片的检测.     

三辛胺萃取稀醋酸过程中各组分的作用

通过实验,研究调节剂对改进混合溶剂热力学平衡特性的作用,阐明应该综合考虑氢键、疏溶作用、分子极性和色散力等分子间相互作用力、溶解度和沸点来选择调节剂.并指出稀释剂在混合溶剂中起着减小粘度、加快传质速度的作用,改进了萃取剂的动力学特性.     

荧光光谱法研究辛烷磺酸钠与BSA的相互作用

用荧光猝灭法研究了水溶液中辛烷磺酸钠（SOS）与牛血清白蛋白（BSA）的相互结合反应．结果表明,SOS与BSA的相互结合作用为动态猝灭．在水溶液中SOS与蛋白质牢固结合,其结合常数K=4．06×10^3．根据Fǒrster非辐射能量转移理论,计算了给体与受体间距离r=7．56nm和能量转移效率E＝0．317,并根据热力学参数推测了SOS与BSA的作用力类型及其随BSA浓度的变化．结合红外和溶液中粒径大小的变化初步探讨了BSA构象的变化．     

模拟水解酶的合成及其催化动力学

介绍了2种具有吗啉环的Schiff碱过渡金属配合物的合成和表征,并且配体相同而金属离子不同的2种配合物作为模拟羧酸酯水解酶被用于催化对硝基吡啶甲酸酯（PNPP）水解。研究了催化反应的动力学机理,建立了PNPP催化水解的动力学模型,并计算了相关的动力学和热力学常数。研究结果表明：2种配合物在催化PNPP水解中表现出较好的催化活性;在缓冲溶液中随着溶液pH的增大,PNPP催化水解速率提高;配合物中金属离子极化作用影响PNPP催化水解速率。     

巴氯芬与胃蛋白酶相互作用的光谱研究

采用荧光光谱法和紫外可见吸收光谱法研究了巴氯芬与胃蛋白酶的结合.观测到巴氯芬使胃蛋白酶的最大紫外吸收峰蓝移.由Stern-Voliner方程求得其荧光猝灭类型为静态猝灭,求出结合常数,由热力学参数判断其作用力主要为疏水作用.根据非辐射能量转移理论,胃蛋白酶与巴氯芬之间发生了能量转移,从而使胃蛋白酶发生了荧光猝灭.用红外光谱法研究了巴氯芬对胃蛋白酶的影响,结果表明:巴氯芬改变了胃蛋白酶的二级结构.     

Gelation of particles with short-range attraction

Nanoscale or colloidal particles are important in many realms of science and technology. They can dramatically change the properties of materials, imparting solid-like behaviour to a wide variety of complex fluids. This behaviour arises when particles aggregate to form mesoscopic clusters and networks. The essential component leading to aggregation is an interparticle attraction, which can be generated by many physical and chemical mechanisms. In the limit of irreversible aggregation, infinitely strong interparticle bonds lead to diffusion-limited cluster aggregation (DLCA). This is understood as a purely kinetic phenomenon that can form solid-like gels at arbitrarily low particle volume fraction. Far more important technologically are systems with weaker attractions, where gel formation requires higher volume fractions. Numerous scenarios for gelation have been proposed, including DLCA, kinetic or dynamic arrest, phase separation, percolation and jamming. No consensus has emerged and, despite its ubiquity and significance, gelation is far from understood-even the location of the gelation phase boundary is not agreed on. Here we report experiments showing that gelation of spherical particles with isotropic, short-range attractions is initiated by spinodal decomposition; this thermodynamic instability triggers the formation of density fluctuations, leading to spanning clusters that dynamically arrest to create a gel. This simple picture of gelation does not depend on microscopic system-specific details, and should thus apply broadly to any particle system with short-range attractions. Our results suggest that gelation-often considered a purely kinetic phenomenon-is in fact a direct consequence of equilibrium liquid-gas phase separation. Without exception, we observe gelation in all of our samples predicted by theory and simulation to phase-separate; this suggests that it is phase separation, not percolation, that corresponds to gelation in models for attractive spheres.     

苯噻啶与蛋白质作用特征的热力学研究

用荧光光谱法和紫外－可见吸收光谱法研究了在模拟人体生理条件下，苯噻啶与牛血清白蛋白（BSA）结合反应的特征，发现苯噻啶对BSA有较强的荧光猝灭作用，且苯噻啶的紫外吸收光谱和BSA的荧光发射光谱有一定程度的重叠，由此得出了其结合反应的结合常数、结合位置和结合过程的基本热力学参数。     

苦杏仁苷与人血清白蛋白相互作用的荧光光谱研究

应用荧光光谱法,研究了苦杏仁苷与人血清白蛋白(HSA)在pH=7.40的Tris-HCl缓冲溶液中相互作用的情况.研究表明,苦杏仁苷通过静态机理猝灭了HSA的内源荧光.并计算得到苦杏仁苷与HSA的结合常数和结合位点数.通过热力学数据,推断出苦杏仁苷与人血清白蛋白之间主要靠氢键和范德华力结合.同时采用同步荧光技术考察了苦杏仁苷对HSA构象的影响.     

灰黄霉素与牛血清白蛋白结合反应的荧光光谱研究

用荧光光谱法研究了在模拟人体体液条件下,灰黄霉素与牛血清白蛋白结合反应的荧光光谱和紫外-可见吸收光谱,发现灰黄霉素对牛血清白蛋白有较强的荧光猝灭作用,且其紫外吸收光谱和牛血清白蛋白的荧光发射光谱有一定程度的重叠．由Forste非辐射能量转移理论和热力学公式,得出了结合反应的结合常数、结合位置和结合过程基本热力学性质的变化等．     

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.