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

蛋白质折叠的实验研究中的一个主要困难是无法以非扰动的方式在单个残基的水平上探测局部结构和动力学。 非天然氨基酸（UAA）的遗传掺入显著增强了研究大蛋白质的局部环境的能力，因为定义的光谱标记可以在体内位点特异性地掺入蛋白质中。本提案的研究目标是合成、表征和应用同位素标记的UAA，如4-氰基-L-苯丙氨酸和4-乙炔基-L-苯丙氨酸的同位素变体，以相对非扰动的方式作为具有位点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（NMR），和/或荧光光谱配对，以检查不同的本地蛋白质环境中存在的几个蛋白质系统，包括dematin头片段子域（DHP）和superfolder绿色荧光蛋白（sfGFP）。该建议的教育目标是整合到本科化学课程的建议研究在富兰克林马歇尔学院和指导本科研究生调查蛋白质结构，热力学和动力学。 建议的研究方面将纳入普通化学和物理化学课程。 例如，探测蛋白质结构和展开与各种光谱技术的能力将在物理化学1（热力学和动力学）课程探索。至少有10名本科生将参与这项研究。 本科研究提供了个性化教学的机会，大大有助于学生的智力发展。本科研究生的培训将使他们在未来的努力中取得成功，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.