Ultrafast Biophysical Studies Of Proteins

蛋白质的超快生物物理研究

• 批准号: 6508985
• 负责人: Philip A Anfinrud
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6508985
• 关键词: X ray crystallography; bacterial proteins; binding sites; bioengineering /biomedical engineering; biomedical equipment development; biophysics; chemical association; chemical models; electron density; hemoprotein; laser spectrometry; ligands; molecular dynamics; myoglobin; oxygen tension; photolysis; protein folding; protein structure; structural biology; technology /technique development; time resolved data

Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from the highly ordered structure of its native-folded protein, the detailed mechanism by which it functions has proven elusive. This is because enzymes are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding of how proteins function, it is essential to study this "choreography of life" on the molecular scale. During this reporting period, we have focused on two key areas of technology development: the ability to acquire time-resolved structures of proteins with 150 ps time resolution, and the ability to acquire time-resolved spectra of proteins in crystals with less than 100 fs time resolution. These capabilities allow us to probe protein structural dynamics at an unprecedented level of detail and will help unveil the mechanistic details by which proteins achieve their specific functional goals. Significant milestones have been reached in each of these complementary fronts. For example, in a multinational collaborative effort, we have succeeded in recording a time-resolved structure of carbonmonoxy myoglobin (MbCO) 150 ps after dissociation of a ligand. The model system being studied, MbCO, is a ligand-binding heme protein whose structure and structural evolution effect reversible binding of oxygen as well as discrimination against toxic carbon monoxide. The time-resolved x-ray diffraction pattern reveals the electron density of the protein with atomic (< 2 ?) resolution. The difference electron density map, obtained by subtracting the electron density recorded before and after laser photolysis, reveals correlated positive and negative changes in the vicinity of the active binding site. These changes unveil how the protein responds to ligand dissociation, ligand docking, and ligand expulsion into the surrounding solvent. The data are currently being refined with the assistance of a collaborator, Prof. George Phillips of the University of Wisconsin, in order to model the magnitude and direction of the time-dependent atomic displacements. Our in-house effort to probe protein dynamics spectroscopically has achieved another significant milestone: the ability to record the entire visible-near IR absorbance spectrum of a protein crystal with a single < 100 fs pulse of laser light. This capability, which was first demonstrated in February 20001 and reported at the Biophysical Society Meeting in Boston the same month, paves the way for us to probe protein dynamics in crystals as well as in solution. The unprecedented capabilities of this instrument will allow us to characterize the environment dependence of protein dynamics (e.g., crystal vs. solution) as well as develop photolysis protocols that maximize the yield of photoactivation in protein crystals.

酶促反应表现出显着的选择性和效率，在台式化学反应中很少能够实现。虽然很明显，酶的生化能力源于其天然折叠蛋白的高度有序结构，但其功能的详细机制证明是难以捉摸的。这是因为酶不仅是构成活性位点的静态大分子，如其晶体结构所示。相反，它们是动态分子，其编排的运动可以将底物往返于活性位点的运输，并且可以随着时间的推移调节该位点的活性。为了对蛋白质的功能有一种机械理解，必须在分子量表上研究这种“生命编舞”。在此报告期间，我们专注于技术开发的两个关键领域：具有150 PS时间分辨率的蛋白质的时间分辨结构的能力，以及在晶体中获得时间分辨率低于100 fs的晶体中蛋白质的时间分辨光谱的能力。这些功能使我们能够以前所未有的细节探测蛋白质结构动力学，并将有助于揭示蛋白质实现其特定功能目标的机械细节。在这些互补方面，每个互补的方面都达到了重要的里程碑。例如，在跨国协作努力中，我们成功地记录了配体解离后150 ps的碳氧基肌红蛋白（MBCO）的时间分辨结构。正在研究的模型系统MBCO是一种配体结合血红素蛋白，其结构和结构进化效果可逆结合氧以及对有毒碳一氧化碳的歧视。时间分辨的X射线衍射模式揭示了具有原子（<2？）分辨率的蛋白质的电子密度。通过减去激光光解之前和之后记录的电子密度获得的差异电子密度图显示了活动结合位点附近的正变化和负变化。这些变化揭示了蛋白质如何对配体分离，配体对接和配体驱动到周围溶剂中的反应。目前，数据正在威斯康星大学的合作者乔治·菲利普斯（George Phillips）教授的协助下进行完善，以建模时间依赖性原子位移的大小和方向。我们内部探测蛋白质动力学的内部努力取得了另一个重要的里程碑：记录具有激光光的单个<100 fs脉冲的蛋白质晶体的整个可见性NEAR IR吸光度谱。该能力于20001年2月首次证明，并于同月在波士顿举行的生物物理协会会议上进行了报道，为我们铺平了探测晶体和溶液中蛋白质动力学的道路。该仪器的前所未有的能力将使我们能够表征蛋白质动力学（例如晶体与溶液）的环境依赖性以及开发光解方案，从而最大程度地提高了蛋白质晶体中光激活的产量。