Biophysical Characterization Of Macromolecules

该项目的目的是开发新的和改进现有的生物物理方法，用于表征溶液中的生物大分子，并将这些方法协同应用于蛋白质及其相互作用的研究。采用的实验技术是分析超离心，静态和动态光散射，等温滴定量热法，差示扫描量热法，圆二色谱法，和表面等离子体共振生物传感。为了研究溶液中多蛋白质复合物的可逆形成，我们开发了一种新的多信号分析沉降速度分析超离心。它允许目前多达三种蛋白质组分的沉降系数分布的无标记检测，从而测量多个流体动力学分离的蛋白质复合物的化学计量。我们还分析了结合动力学对沉降时间尺度的影响。进行了研究的原则证明，其次是第一个模型应用到不同的生物系统的相互作用的蛋白质。这些包括纤溶酶原激活物抑制剂（PAI-1）复合物与玻连蛋白的扩展异源缔合的合作研究，这在将玻连蛋白从循环重新定向到细胞外基质的定位中是重要的，在细胞外基质中表现出粘附功能。多信号沉降速度方法已被用来记录占领的两个PAI-1不同的结合位点玻连蛋白作为一个必要的组装的高阶复合物。在T细胞活化后的信号转导复合物的研究的合作应用中，检测到衔接蛋白的延伸的三重蛋白复合物的形成。 本研究将流体力学和热力学沉降技术应用于ArfGAPs的PH结构域、HIV包膜蛋白及其相互作用、NK细胞的相互作用等方面的研究。（自然杀伤）细胞表面受体，疟疾表面蛋白，网格蛋白篮组装，念珠藻素诱导的微管蛋白结构，铁调节蛋白1和2与IRE的相互作用，以及糖酵解酶葡糖脑苷脂酶及其与α突触核蛋白（帕金森病的病原体？的疾病）。我们继续努力合作，将我们的生物物理学方法应用于与帕金森病相关的α突触核蛋白的表达过程的表征。 在光学生物传感领域，在确定表面结合位点的功能异质性的计算方法方面取得了进展。先前引入的模型进行了扩展，以考虑质量传输有限的表面结合。这被应用于抗体-抗原模型系统，随后分析与白细胞与组织蛋白相互作用相关的各种配体的整合素受体，研究炭疽保护性抗原的抗体，以及研究具有炭疽LF蛋白的MAPK激酶。我们还开发了研究小分子与表面固定化核酸和蛋白质结合的技术。第一个应用是研究HIV逆转录酶和人RNA酶H的抑制剂，以帮助筛选潜在药物候选分子的化合物文库，并分析抑制机制。进一步的方法学发展的目的是增加表面捕获的分子的回收率，用于将生物传感器与质谱连接。

The intent of this project is to develop new and to improve existing biophysical methodology for the characterization of biological macromolecules in solution, and to apply these methods collaboratively to the study of proteins and their interactions. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. For the study of reversible formation of multi-protein complexes in solution, we have developed a novel multi-signal analysis for sedimentation velocity analytical ultracentrifugation. It permits the label-free detection of sedimentation coefficient distributions of currently up to three protein components, and thus measure the stoichiometry of multiple hydrodynamically separated protein complexes. We have also analyzed the influence of binding kinetics on the time-scale of sedimentation. Studies were conducted for the proof of principle, followed by first model applications to different biological systems of interacting proteins. These include the collaborative study of the extended hetero-association of plasminogen activator inhibitor (PAI-1) complexes with vitronectin, which is important in redirecting the localization of vitronectin from the circulation to the extracellular matrix where an adhesive function is manifested. The multi-signal sedimentation velocity method has been used to document the occupation of two PAI-1 distinct binding sites on vitronectin as a requisite for the assembly of higher-order complexes. In the collaborative application to the study of a signal transduction complex after T-cell activation, formation of extended triple protein complexes of adaptor proteins was detected. We have collaboratively applied the previously developed techniques hydrodynamic and thermodynamic sedimentation techniques to the study of PH domain of ArfGAPs, the HIV envelope proteins and their interactions, interactions of NK (natural killer) cell surface receptors, malarial surface proteins, clathrin basket assembly, cryptophycin induced tubulin structures, iron regulatory protein 1 and 2 interactions with IREs, and the glycolytic enzyme gucocerebrosidase and its interactions with alpha synuclein (the causative agent in Parkinson?s disease). We have continued our efforts to collaboratively apply our biophysical methodology to the characterization of the filamentation process of alpha synuclein, which is associated with Parkinsons disease. In the area of optical biosensing, progress was made in the computational approach to determine the functional heterogeneity of surface binding sites. The previously introduced model was extended to account for mass transport limited surface binding. This was applied to antibody-antigen model systems, followed by the analysis of integrin receptors to a variety of ligands relevant to leukocyte interaction with tissue proteins, the study of antibodies to anthrax protective antigen, and the study of MAPK-kinases with anthrax LF protein. We have also developed techniques for studying small molecule binding with surface-immobilized nucleic acid and proteins. A first application was the study of inhibitors of HIV reverse transcriptase and human RNAse H to aid in the screening of compound libraries of potential drug candidate molecules, and to analyze the mechanism of the inhibition. A further methodological development is aimed at increasing the recovery of surface captured molecules for interfacing biosensors with mass spectroscopy.