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...

Protein surface hydration is fundamental to its structural stability and flexibility, and water-protein fluctuations are essential to biological function. Here, we report a systematic global mapping of water motions in the hydration layer around a model protein of apomyoglobin in both native and molten globule states. With site-directed mutagenesis, we use intrinsic tryptophan as a local optical probe to scan the protein surface one at a time with single-site specificity. With femtosecond resolution, we examined 16 mutants in two states and observed two types of water-network relaxation with distinct energy and time distributions. The first water motion results from the local collective hydrogen-bond network relaxation and occurs in a few picoseconds. The initial hindered motions, observed in bulk water in femtoseconds, are highly suppressed and drastically slow down due to structured water-network collectivity in the layer. The second water-network relaxation unambiguously results from the lateral cooperative rearrangements in the inner hydration shell and occurs in tens to hundreds of picoseconds. Significantly, this longtime dynamics is the coupled interfacial water-protein motions and is the direct measurement of such cooperative fluctuations. These local protein motions, although highly constrained, are necessary to assist the longtime water-network relaxation. A series of correlations of hydrating water dynamics and coupled fluctuations with local protein's chemical and structural properties were observed. These results are significant and reveal various water behaviors in the hydration layer with wide heterogeneity. We defined a solvation speed and an angular speed to quantify the water-network rigidity and local protein flexibility, respectively. We also observed that the dynamic hydration layer extends to more than 10 A. Finally, from native to molten globule states, the hydration water networks loosen up, and the protein locally becomes more flexible with larger global plasticity and partial unfolding.

Although molecular dynamics simulations have become a useful tool in essentially all fields of chemistry, condensed matter physics, materials science, and biology, there is still a large gap between the time scale which can be reached in molecular dynamics simulations and that observed in experiments. To address the problem, many enhanced sampling methods were introduced, which effectively extend the time scale being approached in simulations. In this perspective, we review a variety of enhanced sampling methods. We first discuss collective-variables-based methods including metadynamics and variationally enhanced sampling. Then, collective variable free methods such as parallel tempering and integrated tempering methods are presented. At last, we conclude with a brief introduction of some newly developed combinatory methods. We summarize in this perspective not only the theoretical background and numerical implementation of these methods but also the new challenges and prospects in the field of the enhanced sampling.

The typical fs-resolved fluorescence transients are shown in Figure 2-

Water motion at protein surfaces is fundamental to protein structure, stability, dynamics, and function. By using intrinsic tryptophans as local optical probes, and with femtosecond resolution, it is possible to probe surface-water motions in the hydration layer. Here, we report our studies of local hydration dynamics at the surface of the enzyme Staphylococcus nuclease using site-specific mutations. From these studies of the WT and four related mutants, which change local charge distribution and structure, we are able to ascertain the contribution to solvation by protein side chains as relatively insignificant. We determined the time scales of hydration to be 3-5 ps and 100 -150 ps. The former is the result of local librational͞rotational motions of water near the surface; the latter is a direct measure of surface hydration assisted by fluctuations of the protein. Experimentally, these hydration dynamics of the WT and the four mutants are also consistent with results of the total dynamic Stokes shifts and fluorescence emission maxima and are correlated with their local charge distribution and structure. We discuss the role of protein fluctuation on the time scale of labile hydration and suggest reexamination of recent molecular dynamics simulations.protein hydration ͉ femtosecond dynamics ͉ protein fluctuation ͉ selective mutation F rom the laboratories of the senior authors of this study (A.H.Z. and D.Z.) (1-11), there has been a series of reports regarding the time and length scales of the water layer around protein surfaces. These studies were for proteins subtilisin Carlsberg (2), monellin (3), phospholipase A 2 (5), melittin (9), and human serum albumin (8, 10). A theoretical model was developed to take into account the exchange with bulk water (4, 12), and the dynamics are consistent with molecular dynamics (MD) simulations of residence times (13-16) on time scales from femtoseconds to picoseconds. Earlier NMR studies have reported hydration dynamics (residence times) in the subnanosecond regime (17-20), but, more recently, a claim has been made that water motions at protein surfaces are ultrafast compared with bulk water, only slowing down by a factor of two to three (21,22). This Ͻ10-ps range would imply that the observed long-time hydration dynamics in tens of picoseconds are due to protein side-chain relaxation (22, 23). In our earlier studies (6), we addressed in detail this issue and the reasons for dominance of hydration dynamics. To quantify the contribution of sidechain motions to total solvation on the time scale of hydration, we must carefully alter the local structure while maintaining the same tryptophan site.In this contribution, we report the effect of mutation (four mutants on three site selections and the WT) on hydration of the enzyme Staphylococcus nuclease (SNase). Fig. 1 shows the x-ray structure of the protein, consisting of three ␣-helices and a five-stranded ␤-barrel with a total of 149 amino acids (24). The only single tryptophan residue (W140) has one edge exposed to the surface ...

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Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are similar to the surface hydration water fluctuations on picosecond time scales. Our molecular dynamics simulations also show the binding interface full of water molecules and nonspecific weak interactions. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase.