CAREER: Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids

蛋白质折叠实验研究的一个主要困难是无法以非扰动的方式在单个残基水平上探索局部结构和动力学。非天然氨基酸(UaAs)的遗传掺入显著提高了研究大型蛋白质局部环境的能力，因为定义的光谱标记可以在体内定位地掺入蛋白质中。这项建议的研究目标是合成、表征和应用同位素标记的尿酸，例如4-氰基-L-苯丙氨酸和4-乙炔-L-苯丙氨酸的同位素变体，以相对无扰动的方式作为具有位置特异性的蛋白质结构和动力学的灵敏光谱探针。这些UAA将与红外(IR)、核磁共振(核磁共振)和/或荧光光谱学配对，以检查存在于几个蛋白质系统中的不同局部蛋白质环境，包括DHP和超文件夹绿色荧光蛋白(SfGFP)。这项提议的教育目标是将拟议的研究整合到富兰克林和马歇尔学院的本科化学课程中，并指导研究蛋白质结构、热力学和动力学的本科生。拟议研究的各个方面将纳入普通化学和物理化学课程。例如，在物理化学1(热力学和动力学)课程中将探索用各种光谱技术探测蛋白质结构和展开的能力。至少有10名本科生将参与这项研究。本科生的研究为个性化教学提供了机会，极大地促进了学生的智力发展。对本科生的培训将为他们在未来的事业中取得成功奠定基础，如研究生院、医学院或其他职业道路。

A major difficulty in experimental studies of protein folding is the inability to probe local structure and dynamics at the level of an individual residue in a non-perturbing fashion. Genetic incorporation of unnatural amino acids (UAAs) significantly enhances the ability to study local environments of large proteins, since defined spectroscopic labels can be incorporated site-specifically into proteins in vivo. The research goal of this proposal is the synthesis, characterization, and application of isotopically labeled UAAs, such as isotopic variants of 4-cyano-L-phenylalanine and 4-ethynyl-L-phenylalanine, to serve as sensitive spectroscopic probes of protein structure and dynamics with site-specificity in a relatively non-perturbing fashion. These UAAs will be paired with infrared (IR), nuclear magnetic resonance (NMR), and/or fluorescence spectroscopy to examine different local protein environments present in several protein systems including the dematin headpiece subdomain (DHP) and superfolder green fluorescent protein (sfGFP).The educational goal of this proposal is the integration of the proposed research into the undergraduate chemistry curriculum at Franklin & Marshall College and to mentor undergraduate research students investigating protein structure, thermodynamics, and kinetics. Aspects of the proposed research will be incorporated into both the general chemistry and physical chemistry courses. For instance, the ability to probe protein structure and unfolding with a variety of spectroscopic techniques will be explored in the Physical Chemistry 1 (Thermodynamics and Kinetics) course. At least ten undergraduate students will be involved in the research. Undergraduate research provides the opportunity for personalized teaching that greatly aids the intellectual development of the students. The training of undergraduate research students will position them for success in their future endeavors such as graduate school, medical school, or other career paths.