Early Stages of Apomyogobin Folding

阿肌红蛋白折叠的早期阶段

• 批准号: 0744607
• 负责人: Heinrich Roder
• 金额: $ 60万
• 依托单位: Institute For Cancer Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-15 至 2013-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0744607&HistoricalAwards=false
• 关键词: Early; Stages; Apomyogobin; Folding

This project is aimed at elucidating the early stages of folding of apomyoglobin (apoMb), an a-helical protein known to fold via a series of partially structured states. The structural and kinetic properties of the conformational changes associated with the transition from the unfolded state to an ensemble of intermediate states populated at acidic pH will be studied by combining continuous-flow fluorescence and quenched-flow H/D exchange measurements on the microsecond time scale with site-directed mutagenesis. Key residues involved in stabilizing early intermediates and transition states in folding will be identified by measuring the thermodynamic and kinetic consequences of alanine substitutions at conserved positions within the protein core. The formation of specific tertiary interactions during folding will be probed by replacing small residues at specific helix-helix docking sites by larger hydrophobic side chains, which will disrupt specific side chain contacts while maintaining non-specific hydrophobic interactions. Specific helix pairing interactions will be observed by using cysteine as an intramolecular quencher of tryptophan fluorescence. A novel quenched-flow hydrogen exchange experiment with a dead-time of 60 ms will provide complementary information on the acquisition of hydrogen-bonded structure during the initial stages of apoMb folding. The results obtained will identify critical residues and interactions (the folding nucleus) involved in directing structure formation during initial stages of folding, and will thus elucidate the sequence determinants of protein folding. The findings will provide a firm experimental framework for developing and testing theoretical and computational models of complex folding reactions involving intermediate states.Advanced rapid mixing techniques and detection methods developed in the principal investigator's laboratory not only represent a unique resource for the protein folding community, but also benefit other areas of research, such as kinetic studies of rapid conformational changes and enzymatic reaction mechanisms. This project offers extensive opportunities for training future scientists in experimental techniques and theoretical concepts, including protein biochemistry, advanced kinetic techniques, optical and NMR spectroscopy, and the principles of protein structure and folding. Although the Fox Chase Cancer Center is not a degree-granting institution, the principal investigator has access to and will train students at all levels, including a) graduate students from the University of Pennsylvania and Temple University; b) graduate students visiting Fox Chase through a partnership with universities in Russia; c) a summer research program for undergraduate students; and (d) the Howard Hughes Medical Institutes Student Scientist Program, which places gifted students from local high schools in Fox Chase labs.

该项目旨在阐明脱辅基肌红蛋白（apoMb）折叠的早期阶段，apoMb是一种已知通过一系列部分结构化状态折叠的α-螺旋蛋白。将通过结合连续流荧光和猝灭流H/D交换测量与定点诱变的微秒时间尺度上的结构和动力学性质的构象变化与从未折叠状态的过渡到在酸性pH值下填充的中间状态的集合进行研究。通过测量蛋白质核心内保守位置处丙氨酸取代的热力学和动力学后果，将确定参与稳定折叠中的早期中间体和过渡态的关键残基。将通过用较大的疏水侧链替换特定螺旋-螺旋对接位点处的小残基来探测折叠期间特异性三级相互作用的形成，这将破坏特异性侧链接触，同时保持非特异性疏水相互作用。通过使用半胱氨酸作为色氨酸荧光的分子内猝灭剂，将观察到特定的螺旋配对相互作用。一种新的猝灭流氢交换实验的死时间为60毫秒将提供补充信息的收购过程中的apoMb折叠的初始阶段的氢键结构。所获得的结果将确定关键的残基和相互作用（折叠核）参与指导结构的形成在折叠的初始阶段，从而阐明蛋白质折叠的序列决定因素。这些发现将为开发和测试涉及中间态的复杂折叠反应的理论和计算模型提供坚实的实验框架。首席研究员实验室开发的先进快速混合技术和检测方法不仅代表了蛋白质折叠社区的独特资源，而且还有利于其他研究领域，例如快速构象变化和酶反应机制的动力学研究。该项目为培养未来的科学家提供了广泛的机会，包括实验技术和理论概念，包括蛋白质生物化学，先进的动力学技术，光学和NMR光谱学，以及蛋白质结构和折叠的原则。虽然福克斯蔡斯癌症中心不是一个授予学位的机构，但主要研究者可以接触并将培训各级学生，包括a）宾夕法尼亚大学和坦普尔大学的研究生; B）通过与俄罗斯大学合作访问福克斯蔡斯的研究生; c）本科生暑期研究计划;以及（d）霍华德休斯医学研究所学生科学家计划，该计划将当地高中的天才学生安置在福克斯蔡斯实验室。