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，这样的反应在台式化学反应中很少实现。虽然酶的生化能力显然源自其天然折叠蛋白的高度有序结构，但其发挥作用的详细机制已被证明难以捉摸。这是因为酶不仅仅是具有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，其精心设计的运动可以控制底物进出活性位点的运输，并可以随着时间的推移调节该位点的活性。为了从机制上理解蛋白质的功能，有必要在分子尺度上研究这种“生命的编排”。在本报告期内，我们重点关注两个关键的技术开发领域：以 150 ps 时间分辨率获取蛋白质时间分辨结构的能力，以及以小于 100 fs 的速度获取晶体中蛋白质时间分辨光谱的能力时间分辨率。这些功能使我们能够以前所未有的细节水平探索蛋白质结构动力学，并将有助于揭示蛋白质实现其特定功能目标的机制细节。这些互补领域中的每一个都已达到了重要的里程碑。例如，在一项跨国合作中，我们成功记录了配体解离后 150 ps 的碳单氧肌红蛋白 (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，其结构和结构进化影响氧的可逆结合以及对有毒一氧化碳的辨别。时间分辨 X 射线衍射图以原子 (< 2 ?) 分辨率揭示了蛋白质的电子密度。通过减去激光光解前后记录的电子密度而获得的差异电子密度图揭示了活性结合位点附近的相关正负变化。这些变化揭示了蛋白质如何响应配体解离、配体对接和配体排出到周围溶剂中。目前正在合作者威斯康星大学的乔治·菲利普斯教授的帮助下对数据进行完善，以便对随时间变化的原子位移的大小和方向进行建模。我们内部通过光谱方式探测蛋白质动力学的努力已经实现了另一个重要的里程碑：能够用单个 < 100 fs 的激光脉冲记录蛋白质晶体的整个可见光-近红外吸收光谱。这种能力于 20001 年 2 月首次得到证实，并于同月在波士顿举行的生物物理学会会议上进行了报告，为我们探索晶体和溶液中的蛋白质动力学铺平了道路。该仪器前所未有的功能将使我们能够表征蛋白质动力学的环境依赖性（例如晶体与溶液），并开发光解方案，最大限度地提高蛋白质晶体中光活化的产量。