Dissection of complex protein dynamics in human thioredoxin

Abstract

We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47-0.67 and 10.8 -13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10 -17.5 ps for three mutants. A robust dynamical process in 95-114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275-615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.active site hydration ͉ femtosecond dynamics ͉ intraprotein electron transfer ͉ sited-directed mutation P rotein dynamics is a complex process and evolves on a multidimensional energy landscape with various interactions and conformations over wide time scales (1-7). To understand such complex dynamics, we need to dissect the process into elementary steps and determine their relevant time scales. Many elementary reactions even occur on a similar time scale (8) and different methods are often integrated to separate each reaction channel and thus elucidate their molecular mechanisms (5, 9-13). Here, we report our direct studies of complex protein dynamics at the active site in human thioredoxin (hTrx). Using site-directed mutagenesis and femtosecond-resolved fluorescence spectroscopy, we break down the complex dynamics into four elementary processes: one solvent relaxation dynamics and three electron-transfer (ET) reactions. Fig. 1 shows the x-ray structure of oxidized hTrx at a 2.1-Å resolution (14). hTrx, an enzyme catalyzing dithiol-disulfide exchanges with substrate proteins, is a compact globular protein with a central core of five strands of ␤-pleated sheet surrounded by four ␣ helices. The active site of hTrx consists of a highly conserved sequence W31-C-G-P-C-K36 that forms a protruding part of the structure between the second ␤ strand and the second ␣ helix. The enzyme contains only a single tryptophan (W31), locate...

Sub-THz Vibrational Spectroscopy of Bacterial Cells and Molecular Components

“…Long relaxation t imes in our experiments are also confi rmed by results fro m recent wo rk using femtosecond-resol ved fluorescence spectroscopy and the other techniques to break down the co mplex p rotein dynamics into four elementary processes and determine their relevant time scales [32,42]. A mong these processes the authors determined "a robust dynamical process in 95-114 ps" that is very close to the relaxat ion time in our experiments with Bru ker spectrometer, and also much longer quenching dynamic processes with time scales of 275-615 ps at a hydrogen bond distance, which can give local fluctuations with v ibrations spectral line width of 0.12-0.054 cm -1 .…”

Mapping hydration dynamics around a protein surface

“…Under this perturbation, the response, in principle, can result from both the surrounding water molecules and the local protein backbone and side chains. However, our recent solvation studies on the enzymes Staphylococcus nuclease (25) and human thioredoxin (26) by systematic mutations of charged and polar residues around the tryptophan probe indicated that the response is dominantly from surfacewater motions and the contributions from the proteins are minor. With the massive data presented here, we are able to examine the global surface hydration dynamics and their relationships to protein properties and its fluctuations.…”

“…These processes represent the dynamic exchange of hydration layer water with outside bulk water via thermal fluctuations. Femtosecond-resolved spectroscopic studies of protein solvation (19)(20)(21)(22)(23)(24)(25)(26) recently have shown the dynamics of surface hydration on picosecond time scales with a biphasic distribution. We attributed the first ultrafast solvation to water local relaxation and the second longer-time dynamics to coupled water-protein fluctuations (25,27).…”

Ultrafast solvation dynamics at binding and active sites of photolyases

“…These dynamics involve local constrained protein and trapped-water motions within angstrom distance and occur on ultrafast time scales (6,7,10,11). Typically, extrinsic dye molecules or synthetic amino acids were used as local optical probes to label function sites, and the local relaxations were observed, ranging from femtoseconds to nanoseconds (5,(12)(13)(14).…”

“…function-site solvation | ultrafast dynamics | spectral tuning | protein rigidity and flexibility | femtosecond-resolved emission spectra D ynamic solvation in binding and active sites plays a critical role in protein recognition and enzyme reaction, and such local motions optimize spatial configurations and minimize energetic pathways (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). These dynamics involve local constrained protein and trapped-water motions within angstrom distance and occur on ultrafast time scales (6,7,10,11).…”