In this project, funded jointly by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Experimental Physical Chemistry Program in the Chemistry Division, the PI will develop Resonance Raman Spectroscopy as a tool to determine the interaction mechanisms, by which functionally relevant asymmetric distortions of the macrocycle of metalloporphyrins are induced. This involves intramolecular interactions with peripheral substituents and intermolecular interactions with a protein environment. Out-of-plane distortions will be determined by measuring the intensity of Raman bands of out-of-plane modes, which is proportional to the square of the displacement along the distortional coordinate. In-plane distortions will be obtained by applying Polarized Resonance Raman Dispersion spectroscopy, which involves the measurement and analysis of the depolarization ratio dispersion of structure sensitive Raman lines. Well-characterized metalloporphyrins will be utilized to correlate the vibronic coupling parameters of out-of-plane and in-plane modes with symmetry classified distortion parameters, so that the former can be used to determine the distortions of porphyrins and heme groups of unknown structures. In addition to Raman measurements, the PI will record the Circular Dichroism of the investigated porphyrins to correlate the rotational strength to the amount of out-of-plane heme distortions. The results of the Raman studies will be used to discriminate between the contributions of electronic and vibronic perturbations to the splitting of bands in the optical spectrum of heme proteins. These studies will clarify to what extent and by which mechanism the latter are determined by a linear Stark effect due to the internal electric field in the heme cavity. All these investigations will be carried out with a set of natural and 'side directed,' functionally well characterized mutants to identify the contributions of specific amino acids to heme distortions.
This project will contribute in general to the structure analysis of porphyrins and heme proteins in that the employed techniques will enable the detection of subtle but functionally relevant structural changes of the heme group in the sub-Angstrom range. This cannot be accomplished by X-ray crystallography. Graduate and undergraduate students will be trained at the interface of chemistry, biochemistry, and molecular biology. They will learn quantum mechanics, theory and practice of Raman, optical absorption and CD spectroscopy, protein biochemistry, and the different aspects of the relationship between structure and function of proteins. Students from underrepresented groups will be involved in the project.
