There is considerable evidence for the slaving of biomolecular dynamics to the motions of the surrounding solvent environment, but to date there have been few direct experimental measurements capable of site-selectively probing both the dynamics of the water and the protein with ultrafast time resolution. Here, two-dimensional infrared spectroscopy (2D-IR) is used to study the ultrafast hydration and protein dynamics sensed by a metal carbonyl vibrational probe covalently attached to the surface of hen egg white lysozyme dissolved in D(2)O/glycerol solutions. Surface labeling provides direct access to the dynamics at the protein-water interface, where both the hydration and the protein dynamics can be observed simultaneously through the vibrational probe's frequency-frequency correlation function. In pure D(2)O, the correlation function shows a fast initial 3 ps decay corresponding to fluctuations of the hydration water, followed by a significant static offset attributed to fluctuations of the protein that are not sampled within the <20 ps experimental window. Adding glycerol increases the bulk solvent viscosity while leaving the protein structurally intact and hydrated. The hydration dynamics exhibit a greater than 3-fold slowdown between 0 and 80% glycerol (v/v), and the contribution from the protein's dynamics is found to slow in a nearly identical fashion. In addition, the magnitude of the dynamic slowdown associated with hydrophobic hydration is directly measured and shows quantitative agreement with predictions from molecular dynamics simulations.

Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and inter-protein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an allencompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semi-empirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository website (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-theart multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy reveals picosecond protein and hydration dynamics of crowded hen egg white lysozyme (HEWL) labeled with a metal-carbonyl vibrational probe covalently attached to a solvent accessible His residue. HEWL is systematically crowded alternatively with polyethylene glycol (PEG) or excess lysozyme in order to distinguish the chemically inert polymer from the complex electrostatic profile of the protein crowder. The results are threefold: (1) A sharp dynamical jamming-like transition is observed in the picosecond protein and hydration dynamics that is attributed to an independent-to-collective hydration transition induced by macromolecular crowding that slows the hydration dynamics up to an order of magnitude relative to bulk water; (2) The interprotein distance at which the transition occurs suggests collective hydration of proteins over distances of 30-40 Å; and (3) Comparing the crowding effects of PEG400 to our previously reported experiments using glycerol exposes fundamental differences between small and macromolecular crowding agents.

This work exploits the passive phase stabilization of diffractive optics to implement heterodyne detection of the complete χ(5) tensor of liquid CS2 as an example of a simple liquid. This approach permits the use of two different colors for the excitation, probe, and detection beam protocols and enables full optimization of the signal with respect to discrimination against lower order cascaded third-order responses. This work extends the previous study of polarization selectivity, in combination with heterodyne detection, to all independent polarization components to provide further insight into the origins of the fifth-order response and its connection to the multitime correlation of the liquid dynamics. The characteristic feature that clearly distinguishes the direct fifth-order response from lower order cascades is the pronounced ridge along the τ4 axis (probe pulse delay) with very rapid decay along the τ2 axis (excitation pulse delay). This observation is in contrast to recent related work using one-color homodyne detection. With the determination of the direct fifth-order and cascaded third-order signal amplitudes made possible by heterodyne detection, this difference can be attributed to cross terms between the direct fifth-order and cascaded third-order terms inherent to homodyne detection under phase matching conditions used to discriminate against cascades. In support of theoretical treatments, the previously predicted enhancement of rephasing pathways for certain polarization components has been observed. However, even for these tensor elements the remarkable feature is the very rapid decay in the nuclear coherence along τ2. The experiment is predicated on the ability of a 2-quantum transition involving the Raman overtone to rephase the nuclear coherence. These findings indicate that the nuclear motions, in the frequency range accessed, are strongly damped and draw into question the validity of the overtone as a viable pathway for rephasing. With the isolation of the direct fifth-order Raman response, new information regarding relaxation and dephasing pathways in liquids can be determined for the highest frequency modes. The results are in very good agreement with a recent finite field molecular dynamics simulation of liquid CS2 with respect to the polarization dependence of signal magnitudes, relative cascade signal amplitudes, and qualitative agreement with respect to the predicted temporal profiles.

The thermodynamic driving forces for protein folding, association and function are often determined by protein-water interactions. With a novel covalently bound labeling approach, we have used sensitive vibrational probes, site-selectively conjugated to two lysozyme variants–in conjunction with ultrafast two-dimensional infrared (2D-IR) spectroscopy–to investigate directly the protein-water interface. By probing alternatively a topologically flat, rigid domain and a flexible domain, we find direct experimental evidence for spatially heterogeneous hydration dynamics. The hydration environment around globular proteins can vary from exhibiting bulk-like hydration dynamics to dynamically constrained water, which results from stifled hydrogen bond switching dynamics near extended hydrophobic surfaces. Furthermore, we leverage preferential solvation exchange to demonstrate that the liberation of dynamically constrained water is a sufficient driving force for protein-surface association reactions. These results provide an intuitive picture of the dynamic aspects of hydrophobic hydration of proteins, illustrating an essential function of water in biological processes.