The unique features of a macromolecule and water as a solvent make the issue of solvation unconventional, with questions about the static versus dynamic nature of hydration and the physics of orientational and translational diffusion at the boundary. For proteins, the hydration shell that covers the surface is critical to the stability of its structure and function. Dynamically speaking, the residence time of water at the surface is a signature of its mobility and binding. With femtosecond time resolution it is possible to unravel the shortest residence times which are key for the description of the hydration layer, static or dynamic. In this article we review these issues guided by experimental studies, from this laboratory, of polar hydration dynamics at the surfaces of two proteins (Subtilisin Carlsberg (SC) and Monellin). The natural probe tryptophan amino acid was used for the interrogation of the dynamics, and for direct comparison we also studied the behavior in bulk watersa complete hydration in 1 ps. We develop a theoretical description of solvation and relate the theory to the experimental observations. In this theoretical approach, we consider the dynamical equilibrium in the hydration shell, defining the rate processes for breaking and making the transient hydrogen bonds, and the effective friction in the layer which is defined by the translational and orientational motions of water molecules. The relationship between the residence time of water molecules and the observed slow component in solvation dynamics is a direct one. For the two proteins studied, we observed a "bimodal decay" for the hydration correlation function, with two primary relaxation times: ultrafast, typically 1 ps or less, and longer, typically 15-40 ps, and both are related to the residence time at the protein surface, depending on the binding energies. We end by making extensions to studies of the denatured state of the protein, random coils, and the biomimetic micelles, and conclude with our thoughts on the relevance of the dynamics of native structures to their functions.
To better understand DNA photodamage, several nucleosides were studied by femtosecond transient absorption spectroscopy. A 263-nm, 150-fs ultraviolet pump pulse excited each nucleoside in aqueous solution, and the subsequent dynamics were followed by transient absorption of a femtosecond continuum pulse at wavelengths between 270 and 700 nm. A transient absorption band with maximum amplitude near 600 nm was detected in protonated guanosine at pH 2. This band decayed in 191 +/- 4 ps in excellent agreement with the known fluorescence lifetime, indicating that it arises from absorption by the lowest excited singlet state. Excited state absorption for guanosine and the other nucleosides at pH 7 was observed in the same spectral region, but decayed on a subpicosecond time scale by internal conversion to the electronic ground state. The cross section for excited state absorption is very weak for all nucleosides studied, making some amount of two-photon ionization of the solvent unavoidable. The excited state lifetimes of Ado, Guo, Cyd, and Thd were determined to be 290, 460, 720, and 540 fs, respectively (uncertainties are +/-40 fs). The decay times are shorter for the purines than for the pyrimidine bases, consistent with their lower propensity for photochemical damage. Following internal conversion, vibrationally highly excited ground state molecules were detected in experiments on Ado and Cyd by hot ground state absorption at ultraviolet wavelengths. The decays are assigned to intermolecular vibrational energy transfer to the solvent. The longest time constant observed for Ado is approximately 2 ps, and we propose that solute-solvent H-bonds are responsible for this fast rate of vibrational cooling. The results show for the first time that excited singlet state dynamics of the DNA bases can be directly studied at room temperature. Like sunscreens that function by light absorption, the bases rapidly convert dangerous electronic energy into heat, and this property is likely to have played a critical role in life's early evolution on earth.
Biological water at the interface of proteins is critical to their equilibrium structures and enzyme function and to phenomena such as molecular recognition and protein-protein interactions. To actually probe the dynamics of water structure at the surface, we must examine the protein itself, without disrupting the native structure, and the ultrafast elementary processes of hydration. Here we report direct study, with femtosecond resolution, of the dynamics of hydration at the surface of the enzyme protein Subtilisin Carlsberg, whose single Trp residue (Trp-113) was used as an intrinsic biological fluorescent probe. For the protein, we observed two well separated dynamical solvation times, 0.8 ps and 38 ps, whereas in bulk water, we obtained 180 fs and 1.1 ps. We also studied a covalently bonded probe at a separation of Ϸ7 Å and observed the near disappearance of the 38-ps component, with solvation being practically complete in (time constant) 1.5 ps. The degree of rigidity of the probe (anisotropy decay) and of the water environment (protein vs. micelle) was also studied. These results show that hydration at the surface is a dynamical process with two general types of trajectories, those that result from weak interactions with the selected surface site, giving rise to bulk-type solvation (Ϸ1 ps), and those that have a stronger interaction, enough to define a rigid water structure, with a solvation time of 38 ps, much slower than that of the bulk. At a distance of Ϸ7 Å from the surface, essentially all trajectories are bulk-type. The theoretical framework for these observations is discussed.W ater is essential for the stability and function of biological macromolecules, proteins and DNA. Hydration plays a major role in the assembly of a protein's structure and dynamics. For example, water molecules around hydrophobic and hydrophilic sites are important to the understanding of the activity of enzyme proteins (see, e.g., refs. 1-3) and are part of the recognition process by other molecules or proteins. The water molecules that make up the hydration shell in the immediate vicinity of the surface are particularly relevant to the function and, in that sense, are termed biological water; this distinction has been discussed clearly by Nandi and Bagchi (4) in relation to dielectric relaxations. The nature of this shell ''layer'' has been the focus of numerous studies both theoretically and experimentally (see refs. 5-12), yet there is no generalized picture of the dynamics at the local molecular level.X-ray crystallography, neutron diffraction, and molecular dynamics studies have shown (5-10) that at protein surfaces, water molecules are site-selective and can be restricted in their motion, even existing in the form of clusters in some cases. For example, neutron diffraction experiments (9) followed by molecular dynamics simulations on carboxymyoglobin (10) revealed that among the 89 water molecules associated with the protein, 4 remain bound during the entire length of the molecular dynamics simulation (50 ps), whereas ...
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