Ultrafast Hydration Dynamics in Melittin Folding and Aggregation: Helix Formation and Tetramer Self-Assembly

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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...

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confidence: 90%

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confidence: 99%

Dissection of complex protein dynamics in human thioredoxin

Wang

et al. 2007

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“…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).…”

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confidence: 99%

Mapping hydration dynamics around a protein surface

Zhang

Wang

Kao

et al. 2007

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302

373

Protein surface hydration is fundamental to its structure and activity. We report here the direct mapping of global hydration dynamics around a protein in its native and molten globular states, using a tryptophan scan by site-specific mutations. With 16 tryptophan mutants and in 29 different positions and states, we observed two robust, distinct water dynamics in the hydration layer on a few (Ϸ1-8 ps) and tens to hundreds of picoseconds (Ϸ20 -200 ps), representing the initial local relaxation and subsequent collective network restructuring, respectively. Both time scales are strongly correlated with protein's structural and chemical properties. These results reveal the intimate relationship between hydration dynamics and protein fluctuations and such biologically relevant water-protein interactions fluctuate on picosecond time scales.femtosecond dynamics ͉ site-directed mutation ͉ tryptophan scan ͉ water-protein fluctuation W ater motion in the hydration layer is central to protein fluctuation, an essential determinant to its structural stability, dynamics, and function (1-9). Protein surface hydration is a longstanding unresolved problem, but recent extensive studies have merged into a cohesive picture: hydration water molecules are not static but dynamic in nature (1, 2, 10-18). NMR studies (13, 14) have revealed water residence times at protein surfaces within the subnanosecond regime, and molecular dynamics (MD) simulations (15-18) have indicated that water stays in the layer on the time scales from femtoseconds to picoseconds. 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). To generalize the global heterogeneous hydration dynamics around protein surfaces, correlate the dynamics with protein local structures and chemical identities, and decipher the molecular mechanism of waterprotein fluctuations, we report here our direct mapping of water motions around a globular protein, apomyoglobin (apoMb), in its two states, native and molten globular, using intrinsic tryptophan residue (W) as a local molecular probe to scan the surface by protein engineering.Myoglobin from sperm whale has eight ␣-helices (A-H) with a total of 153 aa (Fig. 1) (28), and all experiments were done with apoMb by removal of the prosthetic heme group. We carefully designed more than 30 mutants and placed tryptophan one at a time along each helix at the protein surface. After we screened all mutant proteins with their structural content, stability, and excited-state lifetime of tryptophan, only 16 mutants are appropriate for mapping global hydration, as shown in Fig. 1. We used a laser wavelength of 290 nm with a pu...

“…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). Such labeling of bulky dye molecules usually induces significant local perturbations, and direct characterization with intrinsic chromophores in proteins eliminates those interferences and reveals intact environment responses (4,7,(15)(16)(17)(18)(19)(20)(21), as recently examined in green fluorescence proteins (21). We have recently studied a series of flavoproteins using intrinsic flavin molecule as the optical probe (10,22,23) and especially found the important functional role of local solvation in photolyase (10).…”

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confidence: 99%

Ultrafast solvation dynamics at binding and active sites of photolyases

Chang

Guo

Kao

et al. 2010

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Dynamic solvation at binding and active sites is critical to protein recognition and enzyme catalysis. We report here the complete characterization of ultrafast solvation dynamics at the recognition site of photoantenna molecule and at the active site of cofactor/ substrate in enzyme photolyase by examining femtosecondresolved fluorescence dynamics and the entire emission spectra. With direct use of intrinsic antenna and cofactor chromophores, we observed the local environment relaxation on the time scales from a few picoseconds to nearly a nanosecond. Unlike conventional solvation where the Stokes shift is apparent, we observed obvious spectral shape changes with the minor, small, and large spectral shifts in three function sites. These emission profile changes directly reflect the modulation of chromophore's excited states by locally constrained protein and trapped-water collective motions. Such heterogeneous dynamics continuously tune local configurations to optimize photolyase's function through resonance energy transfer from the antenna to the cofactor for energy efficiency and then electron transfer between the cofactor and the substrate for repair of damaged DNA. Such unusual solvation and synergetic dynamics should be general in function sites of proteins.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 (1-11). 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). Such labeling of bulky dye molecules usually induces significant local perturbations, and direct characterization with intrinsic chromophores in proteins eliminates those interferences and reveals intact environment responses (4, 7, 15-21), as recently examined in green fluorescence proteins (21). We have recently studied a series of flavoproteins using intrinsic flavin molecule as the optical probe (10, 22, 23) and especially found the important functional role of local solvation in photolyase (10).Photolyase, a flavoprotein and a photoenzyme, repairs damaged DNA caused by UV irradiation. Two types of structurally similar photolyases are specific for two major UV-induced DNA lesions of cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (24). The CPD photolyase contains two noncovalently bound chromophores, a pterin molecule in the form of methenyltetrahydrofolate (MTHF) in the binding site as the photoantenna for energy efficiency and a fully reduced flavin adenine dinucleotide (FADH − ) at the active site as the catalytic cofactor for repair of d...