Ultrafast protein hydration dynamics probed by intrinsic tryptophan

Elucidation of water-protein interactions and dynamics is essential to the understanding of protein structure, dynamics and function. In this dissertation, we describe a novel methodology developed in our lab to probe the dynamics of water-protein interactions. The natural amino acid tryptophan is employed as an intrinsic optical probe. By integrating site-directed mutagenesis and state-of-the-art femtosecond laser spectroscopy, we are able to monitor the Stokes shift of tryptophan at any specific positions in proteins, one at a time, and the local hydration dynamics around tryptophan can be precisely determined. This dissertation presents several important protein systems we have investigated with this method. These include the membrane protein melittin, drug delivery protein human serum albumin, and the "hydrogen atom" of modern molecular biology, (apo)myoglobin. These protein systems have very different structures and biological functions. However, a robust double exponential hydration dynamics were observed in all these proteins, which represent two types of water relaxation processes. The first timescale is in several picoseconds and results from the initial local collective water network relaxation mainly through libration motions. The second time scale is in tens to hundreds of picoseconds and results from lateral hydration layer restructuring. The second process is strongly coupled with protein structure fluctuations and dynamic exchange between protein hydration water and bulk water. Both time scales are strongly correlated with protein architecture, such as secondary and tertiary structures, neighboring chemical identities, and protein structural flexibilities. These correlations were first evident in the systems of melittin and human serum albumin. Dynamics of hydration water gradually slows down as the melittin structure changes from random coil to alpha-helix to tetramer. While in human serum albumin, solvation dynamics change consistently during protein conformational transitions between multiple functional states. Then these correlations between hydration dynamics and protein properties were investigated in great detail in the protein apomyoglobin. Sixteen tryptophanyl mutants were designed to scan the protein surface. These tryptophans cover very different local protein environments, such as different secondary structures of loop or alpha-helix and different neighboring charged residue distributions. By changing pH to bring the protein to a folded native or a partially folded molten globular state, we were also able to investigate the global transitions of hydration dynamics during protein folding. The results provide clear and rich information of distinct behaviors of hydration water around various protein environments, revealing that hydration water is an integral part of proteins and directly "controls" their structure, dynamics and function.