Acquisition of Biophysical and Computational Instruments for Studies of Proteins and Protein-Ligand Interactions

该提案要求生物物理仪器和计算设备能够对蛋白质折叠和蛋白质配体相互作用的基本方面进行广泛的跨学科研究。参与的研究小组来自华盛顿大学生物分子结构中心和生物化学系、生物结构系和生物工程系。在过去的五年里，计算机能力的急剧增长引领了结构分子生物学一个真正令人兴奋的新时代。现在有希望在蛋白质折叠和蛋白质配体相互作用的水平上理解蛋白质中大量复杂的原子相互作用。然而，要建立足够的计算模型，需要对相关实验系统进行详细的生物物理表征。我们建议采用生物物理和计算相结合的方法来理解蛋白质结构和功能背后的相互作用。光谱学方法将用于详细探讨蛋白质折叠和蛋白质-配体相互作用的动力学和热力学。这些结果将用于开发计算模型，其长期目标是理解连接氨基酸序列，蛋白质三维结构和蛋白质-配体相互作用的规则。本提案中描述的工作有三个主要重点。第一部分的重点是了解蛋白质折叠对氨基酸序列的依赖，使用极其简单的模型蛋白。第二部分的重点是详细了解模型蛋白质配体相互作用：生物素与链霉亲和素的结合。第三部分旨在扩展在第一部分和第二部分中获得的知识，以开发更通用的方法，用于实验和计算表征各种蛋白质配体相互作用，并用于理解被更大的多聚体蛋白质组装所包围的短肽的“限制性折叠”。这三个领域之间的联系不仅仅是概念上的；解决这三个问题所需的生物物理和计算方法是高度重叠的。在每一种情况下，目前可获得的科学问题的范围都受到我们目前无法通过实验确定诸如开率和关率等关键动力学参数以及监测蛋白质折叠反应的限制。我们同样受到当前计算资源的限制，无法模拟模型系统的大小和复杂性。因此，所要求的仪器包括：(1)吸收和圆二色光谱仪，带有相关的止流装置，用于跟踪溶液中蛋白质折叠的热力学和动力学；(2)表面等离子体共振（SPR）装置，用于精确灵敏地测量蛋白质与配体结合的动力学；(3)计算机设备，以支持研究的理论和预测方面以及正在研究的模型系统的可视化。

This proposal requests biophysical instrumentation and computing equipment to enable a broad interdisciplinary investigation of fundamental aspects of protein folding and protein-ligand interactions. The participating research groups are from the Biomolecular Structure Center and the departments of Biochemistry, Biological Structure, and Bioengineering at the University of Washington. The dramatic increase in the power of computers over the last five years has ushered in a truly exciting new era in structural molecular biology. There is now hope of understanding the multitude of complex interatomic interactions in proteins at the level of protein folding and proteinligand interactions. The development of adequate computational models, however, requires detailed biophysical characterization of relevant experimental systems. We propose to take a combined biophysical and computational approach to understanding the interactions which underlie the structure and function of proteins. Spectroscopic methods will be used to probe the kinetics and thermodynamics of protein folding and protein-ligand interactions in detail. These results will be used to develop computational models that have as their long range goals the understanding of the rules linking amino acid sequences, protein 3-dimensional structures, and protein-ligand interactions. The work described in this proposal has three main thrusts. The first part is focused on understanding the dependence of protein folding on amino acid sequence, using extremely simple model proteins. The second part is focused on the detailed understanding of a model proteinligand interaction: the binding of biotin to streptavidin. The third part seeks to extend knowledge gained in parts one and two to develop more general methods for experimentally and computationally charactelizing a wide valiety of protein ligand interactions, and for understanding the "restricted folding" of a short peptide which is enclosed by a larger multimeri c protein assembly. The links between three areas are not merely conceptual; the biophysical and computational methods required to address all three issues are highly overlapping. In each case the range of scientific questions currently accessible is limited by our current inability to experimentally determine such key kinetic parameters as on-rates and off-rates and to monitor protein folding reactions. We are similarly limited by current computational resources as to the size and complexity of model systems which may be simulated. The requested instrumentation therefore consists of (1) absorption and circular dichroism spectrometers with associated stopped-flow apparatus to follow the thermodynamics and kinetics of protein folding in solution, (2) surface plasmon resonance (SPR) apparatus which allows exquisitely sensitive measurement of the kinetics of protein-ligand binding, and (3) computer equipment to support the theoretical and predictive side of the investigation as well as visualization of the model systems under study.