Folding Mechanisms of Dihydrofolate Reductase and the Response Regulators

这个项目的总体目标是了解蛋白质的氨基酸序列将其快速有效地折叠成天然的、有功能的形式的机制。这些研究的目标将是阿尔法/贝塔/阿尔法类蛋白质结构的几个代表，这是生物学中最常见的主题之一。该实验室先前对这一类中的两个成员--二氢叶酸还原酶(DHFR)和H-ras p21的研究揭示了复杂的动力学过程，这些过程开始于亚毫秒的时间范围，并以平行的路径通过瞬时中间体进行。未来的研究将集中在DHFR和几个黄毒素折叠的代表，包括反应监管机构Chey，NTRC和SPOFF。传统的小角X射线散射仪和在前一个授权期开发的新的时间分辨荧光光谱技术将被用来检测未折叠状态下的非随机结构，并监测DHFR、CHEY、NTRC和SPOFF在微秒时间范围内的折叠反应。基因工程方法将被用来创造适合用荧光团标记的突变蛋白质，这是使用福斯特共振能量转移技术进行距离测量所需的。突变也将被用来测试特定的β链和阿尔法螺旋在未折叠蛋白质中的非随机结构中的参与，并指导折叠的早期阶段。所获得的关于瞬时中间体以及将它们与稳定热力学状态分开的势垒的信息将加强对α/β/α折叠机制的理解，并为模拟折叠反应的理论家提供有用的参数。这一努力的更广泛影响是多方面的。首先，对该研究项目的支持将促进马萨诸塞大学医学院化学生物学研究生项目的发展。用于设计实验、分析数据和解释结果的概念和方法促使生物医学研究生院开设了一门分子生物物理学课程，该课程将教授给一年级和二年级的学生。伍斯特地区的本科生也可以参加这门课程，包括克拉克大学和伍斯特理工学院的本科生。其次，本科生、研究生和博士后研究员将接受分子生物物理学方面的培训，这将有助于推进重要的研究目标，并为他们在工业、学术界和政府的专业职业生涯做好准备。第三，在前一批赠款期间开发的新技术将得到改进并应用于蛋白质折叠问题，这是实现基因组测序努力充分受益的最重要障碍之一。已经提交了一项专利披露，以鼓励将这种微流控混合技术应用于发生微秒反应的其他生物或化学领域。

The overall goal of this project is to understand the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to a native, functional form. The target of these studies will be several representatives of the alpha/beta/alpha class of protein structures, one of the most common motifs found in biology. Prior work from this laboratory on two members of this class, dihydrofolate reductase (DHFR) and H-ras p21, has revealed complex kinetic processes that begin in the sub-millisecond time range and proceed through transient intermediates in parallel pathways. Future studies will focus on DHFR and several representatives of the flavodoxin fold, including the response regulators CheY, NtrC, and SpoOF. Conventional small angle x-ray scattering instrumentation and a novel time-resolved fluorescence spectroscopy technique developed over the previous grant period will be used to detect non-random structure in unfolded states and monitor folding reactions in the microsecond time range for DHFR, CheY, NtrC, and SpoOF. Genetic engineering methods will be used to create mutant proteins suitable for labeling with fluorophores required for distance measurements using Forster resonance energy transfer techniques. Mutations will also be employed to test the involvement of specific beta strands and alpha helices in non-random structure in the unfolded protein and in guiding the early stages of folding. The information obtained on transient intermediates and the barriers separating them from stable thermodynamics states will enhance the understanding of the folding mechanism of the alpha/beta/alpha fold and provide parameters useful to theorists who simulate folding reactions.The broader impact of this effort is multi-fold. First, support of this research project will enhance the development of a graduate program in Chemical Biology at the University of Massachusetts Medical School. The concepts and methods employed to design experiments, analyze the data, and interpret the results have motivated the creation of a course in Molecular Biophysics which will be taught to first and second year students in the Graduate School for Biomedical Sciences. This course is also available to undergraduates in the Worcester area, including those at Clark University and Worcester Polytechnic Institute. Second, undergraduates, graduate students, and postdoctoral fellows will receive training in molecular biophysics which will serve to advance important research goals and to prepare them for professional careers in industry, academia, and government. Third, novel technology developed during the previous grant period will be refined and applied to the protein folding problem, one of the most significant impediments to realizing the full benefit of the genomic sequencing efforts. A patent disclosure has been filed to encourage the application of this micro-fluidics mixing technology to other areas of biology or chemistry where microsecond reactions occur.