Fundamental Processes in the Folding of Helical Proteins

该项目将为蛋白质折叠问题的基本方面提供见解，并将为理论和计算研究提供重要的基准。理论，模拟和实验之间的潜在协同作用已经跨越了对折叠非常迅速的小螺旋蛋白的巨大兴趣。它们是解决折叠过程中基本问题的优秀模型系统，部分原因是它们是所有原子模拟的易于处理的目标，部分原因是它们也是组装更复杂结构的第一步的良好模型。将进行几种螺旋蛋白的实验研究。绒毛头饰螺旋子结构域（HP-36）的折叠一直是众多计算和理论研究的主题，但其折叠机制在实验上几乎一无所知。位点定向诱变将与特定同位素标记和快速动力学技术相结合，以开发HP-36折叠的详细图片。展开态结构在快速折叠中的作用是一个有争议的话题。HP-36的未展开状态包含重要的结构，因此它是解决这个问题的一个很好的模型系统。HP-36展开态的结构将用核磁共振进行表征。将研究改变HP-36变性状态结构的突变的动力学后果。该项目的另一个目标是测试上下文如何改变折叠的速度。有人认为，快速折叠和展开可能具有重要的生物学意义，但迄今为止研究的所有快速折叠蛋白质要么是设计的小蛋白质，要么是较大系统的片段。没有人在生物学上更相关的背景下研究过它们完整的亲本结构。HP-36是较大的绒毛头饰（HP-67）的c端子结构域。将HP-36单独折叠和作为HP-67的一部分折叠进行比较，以确定其快速折叠和展开是否受到环境的影响。该项目的最终目标涉及下坡折叠，即在没有自由能垒的情况下折叠。将进行实验来验证折叠新观点的一个更引人注目的理论预测，即稳定非常快速折叠的蛋白质会导致向下折叠。合理设计的HP-36超稳定变体将用于这些研究。该项目将为研究生和本科生提供蛋白质生物物理学和蛋白质化学方面的广泛跨学科培训。本科生参与和本科课程开发是项目的重要组成部分。我们已经迈出了第一步；大一本科生普通化学课程的内容已被修订，以包括现代生物化学的材料。新的举措将包括开发一个学期的化学生物学和生物物理化学本科课程。

This project will provide insight into fundamental aspects of the protein folding problem and will generate important benchmarks for theoretical and computational studies. The potential synergy between theory, simulation and experiment has spanned a tremendous interest in small helical proteins that fold very rapidly. They are excellent model systems for addressing fundamental issues in the folding process, in part because they are tractable targets for all atom simulations and in part because they are also good models for the first steps in the assembly of more complex structures. Experimental studies of several helical proteins will be undertaken. The folding of the Villin headpiece helical subdomain (HP-36) has been the subject of numerous computational and theoretical studies but virtually nothing is known experimentally about its folding mechanism. Site directed mutagenesis will be combined with the incorporation of specific isotopic labels and rapid kinetic techniques to develop a detailed picture of the folding of HP-36. The role of unfolded state structure in rapid folding is a controversial topic. The unfolded state of HP-36 contains significant structure thus it is an excellent model system for addressing this topic. Structure in the unfolded state of HP-36 will be characterized using NMR. The kinetic consequences of mutations that alter the denatured state structure of HP-36 will be examined. An additional goal of the project is to test how rapid folding is altered by context. Arguments have been made that rapid folding and unfolding could be biologically significant but all fast folding proteins studied to date are either small designed proteins or fragments of larger systems. None have been studied in the more biologically relevant context of their intact parent structures. HP-36 is the C-terminal subdomain of the larger Villin headpiece (HP-67). The folding of HP-36 in isolation and when it is part of HP-67 will be compared in order to determine if its rapid folding and unfolding is affected by context. The final objective of the project concerns downhill folding, i.e. folding in the absence of a free energy barrier. Experiments will be conducted to test one of the more striking theoretical predictions of the new view of folding, namely that stabilizing very fast folding proteins can lead to downhill folding. Rationally designed hyperstable variants of HP-36 will be used in these studies.The project will provide broad interdisciplinary training in protein biophysics and protein chemistry to both graduate and undergraduate students. Undergraduate participation and undergraduate curriculum development are important parts of the project. The first steps have already been taken; the content of the large first year undergraduate general chemistry course has been revised to include material on modern biological chemistry. New initiatives will include the development of a one semester advance undergraduate course in chemical biology and biophysical chemistry.