Biophysical Characterization of Alpha-Synuclein

该项目的长期研究目标是确定a- synuclein（帕金森病路易体的主要蛋白质成分）的氨基酸序列和周围环境的变化如何影响结构、稳定性、自结合和/或聚集以及纤维的形成。在学术环境中，职业发展规划的长期目标是成为蛋白质结构、稳定性、折叠和聚集领域的基础研究人员和科学领导者。特别是，我将集中精力研究蛋白质折叠和错误折叠的决定因素（周围环境和氨基酸序列），以及错误折叠、聚集和沉淀与老年人群中普遍存在的神经系统疾病的关系。在这个问题多于答案的困难领域进行生物物理研究的紧迫性非常强。通过对新技术的掌握，对我已有经验的人的微调，课程作业，技术研讨会，会议演讲，以及整个获奖过程中的协作互动，我将成为一名完整的科学家，能够解决这个领域固有的和不断发展的许多问题和不确定性。重点和一般方法是建立肽模型和表达系统，用于生产和纯化携带a-synuclein和（3-synuclein，一种与a-synuclein高度同源的蛋白质）特异性突变的野生型和变体。在第一个目标中，将比较a-synuclein和(3-synuclein的构象。这个目标的长期目标是确定与a-和3-突触核蛋白的天然构象有关的因素。在第二个目标中，（3-synuclein）不会在vin-o中聚集的假设将得到检验。该目标的长期目标是确定聚集的决定因素（即氨基酸序列的差异，由老化引起的蛋白质修饰，环境因素）并获得有关聚集结构的信息。第三个目的是验证一个假设，即化学或分子伴侣或脂质囊泡的存在会在溶液中诱导a-突触核蛋白的结构，从而抑制不溶性纤维结构的形成。这个目标的长期目标是为路易体的形成提供一种机制。

The long-term research objective of this project is to define how variations in amino acid sequence and surrounding environment of a- synuclein, the major protein component of Lewy bodies in Parkinson Disease affect structure, stability, self-association and/or aggregation, and fibril formation. The long-term goal of the career development plan in the academic setting is to become a fundamental researcher and scientific leader in the field of protein structure, stability, folding and aggregation. In particular, I will concentrate my efforts on the determinants (surrounding environment and amino acid sequence) of folding and misfolding of proteins and the relation of misfolding, aggregation, and precipitation to the neurological disorders which are so prevalent in the aged population. The urgency for biophysical research in this difficult area in which there are more questions than answers is very strong. By the mastering of new techniques, the fine- tuning of others with which I have already had experience, course work, technique workshops, conference presentations, and collaborative interactions throughout the course of the award, I will become complete as a scientist who is competent to address many of the questions and uncertainties which are inherent and continue to evolve in this field. The focus and general approach is to generate peptide models and expression systems for the production and purification of wild-type and variants bearing specific mutations of a-synuclein and (3-synuclein, a protein highly homologous to a-synuclein. In the first aim, the conformations of a-synuclein and (3-synuclein will be compared. The long-term goal of this aim is to determine the factors involved in dictating the native conformations of both a- and (3-synuclein. In the second aim, the hypothesis that (3-synuclein will not aggregate in vin-o will be tested. The long-term goal of this aim is to define the determinants of aggregation (i.e. differences in amino acid sequence, modifications of the protein resulting from aging, environmental factors) and to obtain information on aggregate structure. The third aim will test the hypothesis that the presence of chemical or molecular chaperones, or lipid vesicles that induce structure in a-synuclein in solution will inhibit the formation of insoluble fibrillar structures. The long-term goal of this aim is to provide a mechanism for the formation of Lewy bodies.