Controlling Enzyme Structure and Activity with Light Illumination

摘要提案题目：用光照控制酶的结构和活性提案号：cts - 055415项目负责人：C. Ted Lee, jr .机构：南加州大学分析（决策依据）：酶在活性过程中与多种分子相互作用，导致构象改变到中间折叠状态，影响几乎所有的生物过程。因此，蛋白质的一般形式-功能关系不应该被认为是静态的；相反，要完全理解这种关系，需要了解蛋白质或酶是如何对各种分子做出反应的。要正确地研究这一现象需要两种互补的方法：(1)以受控的方式诱导蛋白质展开的方法，(2)以相对高的分辨率研究未折叠蛋白质的构象的方法。为了实现这一串联目标，本项目将追求两种新技术，即蛋白质与光可逆表面活性剂的相互作用，以及使用小角中子散射（SANS）来确定部分折叠/未折叠酶在光响应表面活性剂下的结构。表面活性剂通过破坏分子内氨基酸的接触来展开蛋白质。在光反应性表面活性剂的情况下，表面活性剂-氨基酸结合可以通过简单的光照打开或关闭，从而实现对蛋白质构象的光控制。然后用SANS实验确定蛋白质构象，通过应用形状重建方法来优化散射中心（即蛋白质氨基酸或单个原子，取决于所需的分辨率）的位置，以便最佳地拟合散射数据。这种结合的方法将允许充分表征酶和蛋白质中光控制的天然中间变性转变，为折叠途径提供独特的见解。通过光控制蛋白质的形态，光控制蛋白质的功能也将实现，允许酶活性的开关与光照明。光控酶折叠至少需要一个普通的商业黑光灯，因此，可以很容易地移植到教室，提供一个简单的协议，无缝地将研究融入课程作业。这将有助于重新连接从实验室到课程的思想流，并将更好地为学生准备迎接技术多样化的现代工作场所的挑战。这些努力将传播给科学界，特别是学位项目的成果，四年研究项目提供横向（加班）和纵向（跨课程）研究和教育的整合。

AbstractProposal Title: Controlling Enzyme Structure and Activity with Light Illumination Proposal Number: CTS-0554115Principal Investigator: C. Ted Lee, Jr.Institution: University of Southern CaliforniaAnalysis (rationale for decision): Enzymes interact with a variety of molecules during the course of activity, resulting in conformation changes to intermediately-folded states that influence nearly all biological processes. Thus, the general form-function relationships of proteins should not be considered static; instead, to fully understand this relationship requires knowledge of how a protein or enzyme unfolds in response to various molecules. To properly investigate this phenomenon requires two complementary approaches: (1) a means to induce protein unfolding in a controlled manner, and (2) a means to study the conformation of unfolded proteins at relatively high resolutions. To achieve this tandem goal, two novel techniques will be pursued in this project, namely the interaction of proteins with photoreversible surfactants, and the use of small-angle neutron scattering (SANS) to determine the structure of partially-folded/unfolded enzymes in response to photoresponsive surfactants. Surfactants unfold proteins by disrupting intramolecular amino acid contacts. In the case of photoresponsive surfactants, the surfactant-amino acid associations can be switched on or off with simple light illumination, resulting in photocontrol of protein conformation. Protein conformation will then be determined with SANS experiments by applying shape-reconstruction methods to optimize the positions of scattering centers (i.e., protein amino acids or individual atoms, depending on the desired resolution) in order to best fit the scattering data. This combined approach will allow full characterization of the photocontrolled native intermediate denatured transitions in enzymes and proteins, providing unique insight into folding pathways. By controlling protein form with light, photocontrol of protein function will also be achieved, allowing enzymatic activity to be switched on and off with light illumination.Photo-controlled enzyme folding requires at a minimum nothing more than a common, commercial black light and, as such, can be easily ported to the classroom to provide an uncomplicated protocol to seamlessly blend research into coursework. This will serve to reconnect the flow of ideas from the laboratory to the curriculum, and will better prepare students to meet the challenges of a technically diverse, modern day workplace. These efforts will be disseminated to the scientific community, particularly the results of Degree Projects, four-year research projects that provide horizontal (over-time) and vertical (cross-course) integration of research and education.