Protein Surface Hydration Dynamics Via Ultrafast Fluorescence Spectroscopy

Almost all protiens can only perform their biological function or activity in an aqueous environment, either on cellular or molecular scale. Protein hydration is vitally improtant for their structural stability, flexibility, dynamics and biological functions. Thus, it is significant to study the process of protein hydration dynamics in order to understand the biological functions of the protein. However, spatial and temporal resolutions make technical problems in the exploration of protein hydration dynamics. Protein molecular dynamics project is charecterized by a wide distribution of time scale, including ultrafast dynamic processes of chemical bond fluctuation and solvent relaxation, the moderately slower process of protein flip and the much slower process of protein folding. To detect all the activities above, a probe with large time scale, excellent resolution and high sensitivity to the local environment is urgently in need. Tryptophan is one of the three naturally encoded amino acids, featuring high quantum yield, relatively longer fluorescence emission wavelength and sensitive detection for local environment within 10A via dipole-dipole/charge reaction. Besides, to keep the original structure of the protein molecule as far as possible through site-directed mutations, the intrinsic naturally encoded tryptophan makes an ideal probe for the exploration of protein surface hydration.In this thesis, with the help of site-directed mutations, we resolve to perform systematic research on the protein hydration dynamics by a home-built ultrafast fluorescence spectrometer. Using tryptophan as the local probe after site-directed mutations, we studied the protein surface hydration of Staphylococcus nuclease (SNase) and the projection of intracellular interaction dynamics of dipeptide Trp2 in aqueous solution. For the experimental instruments, we have built a femtosecond upconversion fluorescence system and picosecond time-correlated single photon counting (TCSPC) spectrometer, with instrument response functions (IRF) of 330 fs and 550 ps, respectively. The IRF of upconversion system is determined by cross-correlation signal between the Raman signal (at 328 nm) of water and the gate detection pulse (at 800 nm) in the same experimental condition, while the one of TCSPC setup is measured by the Raley scattering of silicon dioxide nanoparticles under the same condition. Two molecular systems have been designed for our research. One is a protein molecular system centring SNase and its mutants. In the SNase protein system, there are three charged residues (K110, E129 and K133) around tryptophan W140 within the distance of 10A. In this study, we use an alanine (A) scan to further mutate any of two charged residues each time and finally replace all three neighboring charged residues to have a hydrophobic environment. The other is a dipipetide molecular system containing Trp2, its derivants N-(tert-Butoxycarbonyl)-L-tryptophan-L-tryptophan (NBTrp2), L-tryptophan-L-tryptophan methyl ester (Trp2Me), and N-acetyl-L-tryptophan-L-tryptophan methyl ester (NATrp2Me).Two dynamics processes at picosecond scale, which evoked heated discussions, have been found in SNase molecular system in our study. Despite other researchers’idea that they are caused by charged side chains of the protein, we believe this motion is induced by water molecule. Since there is only one tryptophan residue W140 in SNase molecular system, we systemically mutated three charged residues around tryptophan to ascertain the cause of the motion and to understand the molecular origin of ultrafast water-protein coupled interactions. When the charges carried by the residues adjacent to tryptophan, the total Stokes shift increases slightly while the charged side chains are not sensitive to relaxation dynamics process. The total Stokes shifts are dominantly from hydration water relaxation and the slow dynamics is from water-driven relaxation, coupled to local protein fluctuations, that is, the hydration relaxations drive the side chain motions. In one word, there are two kinds of protein-water coupled process at picosecond scale. One is the process of regional relaxation of water network, performing in several picoseconds. The other is the interaction between water and protein, i.e. the regroup of water network, which happens in tens to hundreds picosecond, with vital importance to many biological functions such as ligand recogniton and enzyme catalysis.In the dipipetide study, mutiple dynamic processes appear in the aqueous solution of Trp2 and its derivants, including water solvent relaxation around 4 ps and a dynamics process around 100 ps. The slower one may result from the joint effort of intramolecular interaction and molecular charge transfer. The molecular dynamics (kd+ki)-1 should consist of Trp residue dynamics kd-1 and intramolecular interaction dynamics ki-1 between two Trp residues. We calculated the intermolecular interaction dynamics times of Trp2, NBTrp2, Trp2Me and NATrp2Me as 3.64 ns,0.93 ns,11.52 ns and 2.40 ns, respectively.