Determination of protein structures by two dimensional vibrational spectroscopy isotope dilution experiments

Prior to this work two-dimensional vibrational echo spectroscopy (2D-IR) had been limited to the determination of small peptide structures. This thesis proposes and demonstrates a methodology that allows the extension of the spectroscopic principles gained with small molecules to the determination of structures of more complex systems, in particular, transmembrane helix dimers. Micelle suspensions of pairs of transmembrane domains are prepared with varying levels of heavy (13C=18O) isotope substitutions in the helix peptide backbone at selected locations. Vibrational coupling causes tertiary delocalization of the vibrational excitation and a splitting of the isotopic pair spectral transitions. The ultrafast time resolution of 2D-IR allows one to determine the vibrational frequency correlation of the selected modes and establish a dynamical model of spectra that is adequate to extract vibrational coupling constants from the comparison of spectra at different isotopic concentrations. The frequency correlation function is also capable of indicating the presence of mobile water associated with the labeled residues. The constraints derived from vibrational coupling of the precisely spaced heavy residues leads to determination of an optimized structure from a range of model candidates. The methods developed herein are applicable and transferable to other protein samples. This approach, as described, will identify conformational exchange or any other dynamical phenomena in addition to characterizing the structure of the exchanging species in picoseconds (ps) or longer timescales.