In this CAREER project funded by the Chemical Structure, Dynamics and Mechanisms Program of the Chemistry Division, Prof. Casey Londergan and a group of undergraduate research assistants at Haverford College will use infrared and Raman spectroscopy, coupled with site-specific chemical labeling schemes, to develop several new methods for spectroscopically isolating specific protein residues and observing changes in their local environments associated with functionally important structural and dynamic changes. Functional groups with unique vibrational frequencies are needed to harness the fast intrinsic time scale of vibrational spectroscopy to report on the entire dynamic conformational distribution around particular sites in proteins with complicated self-assembly and binding activity. This project will develop three such functional groups for use in particular structural contexts, with a dual emphasis on understanding the fundamental physical mechanisms of their environmental dependence and using demonstrated dependences to solve particularly difficult protein-based structural problems. In some contexts, artificial amino acids are most useful, and in others, maintaining natively structured residues is centrally important. The interfacial orientation of membrane-bound proteins will be investigated using the CN stretching band of cyanylated cysteine and the NNN asymmetric stretching vibration of azido-substituted amino acids. The C-D stretching vibration of 2-deuterated histidine will be used to reveal the protonation state and structural role of specific histidine residues in proteins with pH-dependent structural changes.
With a set of physically clear baseline results in hand, this group of new vibrational probe groups will be used to investigate the dynamic structures of proteins associated with neurodegenerative disease and viral infectivity. Undergraduate students of all levels involved in this research will be trained broadly in spectroscopy, chemical manipulation, and protein expression and purification. The integrative research approach will be accompanied by an effort to develop experimental design- and inquiry-based modules for undergraduate teaching laboratories in collaboration with Philadelphia-area high school teachers.
