Fundamental Processes in the Folding of Helical Proteins

这个项目将提供对蛋白质折叠问题的基本方面的洞察，并将为理论和计算研究产生重要的基准。理论、模拟和实验之间的潜在协同作用引起了人们对折叠速度非常快的小螺旋蛋白质的极大兴趣。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容易处理的目标，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。将对几种螺旋蛋白质进行实验研究。维林头饰螺旋结构域(HP-36)的折叠一直是许多计算和理论研究的主题，但几乎对其折叠机制在实验上知之甚少。定点突变将与特定的同位素标记和快速动力学技术相结合，以开发HP-36折叠的详细图像。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的展开状态包含了重要的结构，因此它是解决这一问题的一个很好的模型系统。HP-36在展开状态下的结构将使用核磁共振进行表征。将检查改变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折叠的速度如何随着环境的变化而变化。有观点认为，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小的设计蛋白质，要么是更大系统的片段。没有一个是在其完整的母体结构的更具生物学相关性的背景下进行研究的。HP-36是较大的维林头饰(HP-67)的C末端亚域。将比较HP-36单独折叠和何时成为HP-67的一部分，以确定其快速折叠和展开是否受环境的影响。该项目的最终目标是下坡折叠，即在没有自由能垒的情况下折叠。实验将被用来检验新的折叠观的一个更引人注目的理论预测，即稳定非常快的折叠蛋白质可以导致下坡折叠。这些研究将使用合理设计的HP-36超稳定变种。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的广泛跨学科培训。本科生参与和本科生课程开发是该项目的重要组成部分。已经迈出了第一步；第一年本科普通化学大课的内容已经修改，纳入了现代生物化学的材料。新的举措将包括开发一学期的化学生物学和生物物理化学高级本科课程。

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.