Abstract

Proteins are complex organic compounds that are among the fundamental molecules of biology. They are the building blocks of life on Earth, responsible for most of the complex functions, evolving and adjusting themselves for millions of years of changing condition of the environment. For the last century scientists all around the world are trying to crack down the mystery of protein folding, its structure, sequences, and conformational changes.[1] The advances in different research techniques have uncovered the interplay of these hidden features in many areas, yet we are far away of complete understanding of the functioning of the proteins. Even if today’s methods are capable of providing us with the crystal structure or decipher the amino acid sequence of the protein, the conformational changes affecting the functionality need to be studied further. In order to reveal some parts of this mystery we carry out our research performing Spectral Hole Burning to study protein dynamics. Unfortunately protein does not absorb in the visible range, forcing one to use a dye marker or to pick a protein that naturally has a marker. Presently pigment-protein complexes of photosynthesis process have become a natural choice for such studies, providing necessary information to study the conformational changes associated with light absorption. In this study we focus on Non-Photochemical Spectral Hole Burning (NPHB) and spectral hole recovery experiments on cytochrome b6f protein This protein exhibits behavior that is almost independent of the deuteration of the buffer / glycerol glassy matrix containing the protein, apart from some differences in heat dissipation. On the other hand, strong dependence of the hole burning properties on sample preparation procedures was observed and attributed to a large increase of the electron-phonon coupling and shortening of the excited-state lifetime occurring when n-dodecyl β-D-maltoside (DM) is used as a detergent instead of n-octyl β-D-glucopiranoside (OGP). The data was analyzed assuming that the tunneling parameter distribution or barrier distribution probed by NPHB and encoded into the spectral holes contains contributions from two non-identical components with accidentally degenerate excited state tunneling parameter (hole burning yield) distributions. Both components likely reflect protein dynamics, although with some small probability one of them (with larger mass of the tunneling entity or the displacement along the generalized coordinate) may still represent the dynamics involving specifically the –OH groups of the water / glycerol solvent. Single proton tunneling in the water / glycerol solvent environment or in the protein can be safely excluded as the origin of observed NPHB and hole recovery dynamics. The illumination intensity dependence of the hole growth kinetics in deuterated samples likely reflects differences in heat dissipation between protonated and deuterated samples. These differences are most probably due to the higher interface thermal resistivity between (still protonated) protein and deuterated water / glycerol outside environment.