Biophysical Characterization of Alpha-Synuclein

本项目的长期研究目标是确定α-突触核蛋白（帕金森病中路易体的主要蛋白质组分）的氨基酸序列和周围环境的变化如何影响结构、稳定性、自缔合和/或聚集以及原纤维形成。在学术环境中职业发展计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，我将集中我的努力的决定因素（周围环境和氨基酸序列）的折叠和错误折叠的蛋白质和关系的错误折叠，聚集和沉淀的神经系统疾病是如此普遍的老年人口。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。通过掌握新技术，对我已经有经验的其他技术进行微调，课程工作，技术研讨会，会议演示以及整个奖项过程中的合作互动，我将成为一名能够解决许多问题和不确定性的科学家，这些问题和不确定性是固有的，并将在该领域继续发展。重点和一般方法是产生用于产生和纯化野生型和携带α-突触核蛋白和β-突触核蛋白（一种与α-突触核蛋白高度同源的蛋白质）的特异性突变的变体的肽模型和表达系统。在第一个目标中，将比较α-突触核蛋白和β-突触核蛋白的构象。这个目标的长期目标是确定参与决定α-和β-突触核蛋白的天然构象的因素。在第二个目的中，将检验β-突触核蛋白在vin-o中不聚集的假设。这一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、老化引起的蛋白质修饰、环境因素），并获得关于聚集结构的信息。第三个目标将测试以下假设：在溶液中诱导α-突触核蛋白结构的化学或分子伴侣或脂质囊泡的存在将抑制不溶性纤维状结构的形成。这一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.