Spectroscopic Study of Protein Folding Dynamics

蛋白质折叠动力学的光谱研究

• 批准号: 6657237
• 负责人: FENG GAI
• 金额: $ 5.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6657237
• 关键词: bioengineering /biomedical engineering; biomedical equipment development; conformation; green fluorescent proteins; infrared spectrometry; peptides; protein folding; protein protein interaction; protein sequence; protein structure; structural biology; technology /technique development

DESCRIPTION (provided by applicant): Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Although a great deal of work has been done on the investigation of how the secondary and tertiary structures of proteins are formed, and both experimental and theoretical techniques for studying protein folding are continually becoming more refined, a quantitative and predictive understanding of protein folding is still not attainable. There are still many fundamental questions as to how specific and nonspecific interactions determine the protein folding pathways, the native and nonnative structures, and thermally and kinetically accessible conformation substates, and on what range of timescales do particular folding events occur. Addressing these questions presents the need for further studies with time-resolved spectroscopic techniques that can provide the necessary time resolution and structure sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformation probes that can be used to generate detailed structure interpretations of the transient folding species and their dynamics over the time range of interest. A detailed set of experiments are planned to gain detailed insight into the understanding of various aspects of the folding problem, including the helix-coil transition, early folding events and intermediates, and the mechanism of beta-sheet formation. The technical goals are: (a) to develop a nanosecond temperature jump infrared spectrometer that can measure both transient kinetics at discrete frequencies and time-resolved spectra at discrete reaction times; (b) to develop a microsecond FTIR coupled continuous-flow mixing apparatus; (c) to explore novel isotope editing techniques to permit site specific conformation studies; (d) to introduce nitriles into various amino acids as infrared probes of protein folding, dynamics and interactions; (e) to study the helix-coil transition; (f) to study the beta-sheet formation in the WW domain and its interaction with peptides; (g) to study the folding and spontaneous fluctuation of single GFP molecules.

描述(申请人提供)：蛋白质通过其线性多肽链的特定折叠获得其独特的功能。错误折叠会导致许多疾病，如囊性纤维化和各种神经退行性疾病。尽管人们在研究蛋白质的二级和三级结构是如何形成的方面做了大量的工作，研究蛋白质折叠的实验和理论技术也在不断完善，但对蛋白质折叠的定量和预测理解仍然是不可能的。关于特定的和非特定的相互作用如何决定蛋白质的折叠途径，天然和非天然的结构，以及热和动力学上可获得的构象亚态，以及特定的折叠事件发生在什么时间尺度上，仍然有许多基本的问题。解决这些问题需要进一步研究时间分辨光谱技术，以提供必要的时间分辨率和结构敏感性。因此，拟议研究的主要目标是开发新的光谱方法和新的构象探测器，可用于产生对瞬时折叠物种及其在感兴趣的时间范围内的动力学的详细结构解释。计划进行一系列详细的实验，以详细了解折叠问题的各个方面，包括螺旋-螺旋转变、早期折叠事件和中间体，以及β-折叠形成的机制。技术目标是：(A)开发一种纳秒温度跳跃红外光谱仪，它可以测量离散频率下的瞬时动力学和离散反应时间下的时间分辨光谱；(B)开发微秒FTIR耦合连续流动混合装置；(C)探索新的同位素编辑技术，以便进行特定部位的构象研究；(D)将腈引入各种氨基酸中，作为蛋白质折叠、动力学和相互作用的红外探针；(E)研究螺旋-螺旋转变；(F)研究WW结构域中β-折叠的形成及其与多肽的相互作用；(G)研究单个绿色荧光蛋白分子的折叠和自发涨落。