Dynamics and mechanism of ultrafast water–protein interactions

Abstract

Protein hydration is essential to its structure, dynamics, and function, but water-protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/sidechain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein sidechain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship.hydration shell dynamics | protein side-chain motion | water-driven relaxation | coupled fluctuation | tryptophan scan W ater-protein interactions are critical to protein structural stability and flexibility, functional dynamics, and biological activities (1, 2). Various methods such as neutron scattering (3), NMR (4), laser spectroscopy (5, 6), and molecular dynamics (MD) simulations (7) have been used to reveal protein surface hydration and coupled water-protein dynamics on different time and length scales. Hydration water molecules often participate in various protein functions and their motions even directly "control" protein fluctuations (2, 8). Frauenfelder et al. recently proposed a unified model for protein dynamics (8): large-scale protein motions are slaved to the fluctuations of bulk solvent and controlled by solvent viscosity while internal protein motions are slaved to the fluctuations of the hydration shell and controlled by hydration water. However, direct measurements of such coupled fluctuations at physiological temperature are challenging as a result of the ultrafast nature of water motions, and therefore most studies are indirect or at low temperature (3, 4). Here, we used a tryptophan (W) scan to probe global surface hydration (9) and used femtosecond spectroscopy to follow hydration water motions and local side-chain fluctuations in real time. With temperature dependence, we systematically measured their dynamics and thus finally elucidate their ultimate relationship. Results and DiscussionTryptophan Scan and Femtosecond Fluorescence Spectroscopy. We used DNA polymerase IV (Dpo4) (10), a hyperthermal enzyme without a single tryptophan residue, as a model protein, and designed 10 tryptophan mutants, one at a time, to probe four different domains (Fig. 1A). We performed systematic measurements of tryptophan fluorescence intensity changes with wavelength and time and thereby constructed 3D fluorescence profiles (SI Appendix, SI Note 1). One example of an R176W mutant is ...

How the hydrophobic factor drives protein folding

“…One of the four partly hydrophobic solutes studied by Qvist and Halle (17) was N-acetyl-leucine-N-methylamide, which should have an alkane-like hydration shell. See also the recent NMR study of protein hydration shells by Zhong and coworkers (18).…”

Untangling complex dynamics of biological water at protein–water interface

“…Several additional important results have emerged from the studies reported in ref. 6, which deserve further discussion and scrutiny. First, dynamics probed are sensitive not only to the exposure of the Trp residue but also to the polar character of the surrounding medium.…”

“…These have been derived from experiments that use different techniques, different probes, and different protein molecules (3)(4)(5). The paper by Qin et al (6) discusses results of detailed experimental (and simulation) studies of water at the surface of a specific protein, DNA polymerase IV (Protein Data Bank ID code DPo4). The experimental method used is time-dependent fluorescence Stokes shift (TDFSS), which is a popular tool in the study of dipolar solvation dynamics (1).…”

“…The complexity of a protein-water interface, with its narrow width (only a few nanometers thick), makes a quantitative study, and understanding of its structure and dynamics a challenging task (6)(7)(8)(9)(10)(11). In addition, a protein surface is highly heterogeneous, with a mixture of seemingly randomly placed amino acid residues of diverse polar character.…”

“…Water molecules close to the protein therefore experience different environments from point to point as we travel along the protein surface. As mentioned, Qin et al used TDFSS, using the amino acid residue tryptophan (Trp) as a natural probe (6). By using mutation of the wild variety, the authors placed the probe Trp at different locations on the protein.…”