RUI: Rigorous physical interpretation of vibrational probe frequencies in proteins

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Understanding the structures of proteins and how these structures change in response to their surroundings is a challenge of broad relevance to biology, biochemistry, biomedical science, and disease treatment. Chemists often use optical spectrometers (instruments that measure how molecules absorb, emit or scatter light)to obtain information about molecular structure. But as proteins are large molecules (hundreds or thousands of atoms) that are also flexible, it is very difficult to determine their structures from optical spectroscopy alone. In this project, supported by the Chemical Structure Dynamics and Mechanism (CSDM-A) program of the Chemistry Division, Professor Casey Londergan of Haverford College is using a combination of experimental spectroscopy and computational chemistry to develop "vibrational probe" techniques for the determination of the structures and internal motions of proteins. A vibrational probe is a small molecule that vibrates at a known frequency (typically corresponding to infrared light) when it is isolated, but exhibits a change in frequency when it interacts with other molecules (including other probes). One such probe being investigated is thiocyanate, which contains sulfur, carbon and nitrogen (SCN). By attaching SCN or other vibrational probes to different parts of the protein, Prof. Londergan hypothesizes that the changes in probe frequencies can be associated with changes in their local probe environment. In principle, the vibrational probes thus report on local regions of the proteins and their interactions with other parts of the protein or with other molecules (water, dissolved ions, etc.). However, in order to interpret the experimentally observed probe frequency changes, computer simulations of the different protein shapes and local environments is necessary. This project is being conducted mainly by undergraduate students and is providing them with an interdisciplinary research experience with a network of local and international collaborators. This project also includes educational outreach activities, for example dance-based workshops and instructions designed to engender a more human-level understanding of proteins and their interactions. This research project is focusing on a model regulatory protein (calmodulin), a model membrane protein (alpha-synuclein), and other proteins at the center of bacterial biosynthesis. Well-sampled molecular dynamics (MD) simulations are employed to propose ensembles of structures, and then further simulations with vibrational probe groups explicitly included to produce simulated spectral lineshapes that can validate or help to re-weight the simulated structural ensembles. The initial model associations between measured spectra and local probe environments are being developed based on the relatively broadly-used SCN probe group. As the methodology improves in accuracy, it will ultimately be used to provide quantitative interpretation of new data from less-used probe groups that this project will develop (e.g.,azido, isonitrile, alkyne, and nitro groups). Some of these novel probe groups' signals are more easily collected by Raman spectroscopy rather than infrared absorption. This project's development of a quantitative vibrational probe methodology is likely to enable solutions to many important biomolecular problems where current data are underdetermined for representing the conformational distribution, which is of central importance in disordered and "fuzzy" protein systems(most of which are regulatory and/or disease-related species).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.