Early Stages of Apomyogobin Folding

该项目旨在阐明apoMb折叠的早期阶段，apoMb是一种已知的通过一系列部分结构状态折叠的a-螺旋蛋白质。通过将微秒时间尺度上的连续流动荧光和猝灭流动H/D交换测量与定点突变相结合，将研究在酸性pH下从未折叠状态到中间状态集合的转变相关的构象变化的结构和动力学性质。通过测量蛋白质核心保守位置上丙氨酸取代的热力学和动力学后果，将确定稳定折叠中早期中间体和过渡态的关键残基。在折叠过程中，将通过用更大的疏水侧链取代特定螺旋-螺旋对接位置的小残基来探索特定三级相互作用的形成，这将破坏特定的侧链接触，同时保持非特定的疏水相互作用。用半胱氨酸作为色氨酸荧光的分子内猝灭剂，可以观察到特定的螺旋配对相互作用。一种新的停滞时间为60ms的熄灭流动氢交换实验将为apoMb折叠的初始阶段获得氢键结构提供补充信息。所获得的结果将确定在折叠的初始阶段指导结构形成的关键残基和相互作用(折叠核)，从而将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为蛋白质折叠社区提供了独特的资源，而且有助于其他领域的研究，如快速构象变化和酶反应机理的动力学研究。这个项目为培训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、光学和核磁共振光谱以及蛋白质结构和折叠的原理。虽然福克斯大通癌症中心不是授予学位的机构，但首席研究员可以接触到并将培训各级学生，包括a)宾夕法尼亚大学和坦普尔大学的研究生；b)通过与俄罗斯大学的合作访问福克斯大通的研究生；c)面向本科生的暑期研究项目；以及(D)霍华德·休斯医学院学生科学家计划，该计划将当地高中的天才学生安置在福克斯大通实验室。

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.