The focus in protein folding has been very much on the protein backbone and sidechains. However, hydration waters make comparable contributions to the structure and energy of proteins. The coupling between fast hydration dynamics and protein dynamics is considered to play an important role in protein folding. Fundamental questions of protein hydration include, how far out into the solvent does the influence of the biomolecule reach, how is the water affected, and how are the properties of the hydration water influenced by the separation between protein molecules in solution? We show here that Terahertz spectroscopy directly probes such solvation dynamics around proteins, and determines the width of the dynamical hydration layer. We also investigate the dependence of solvation dynamics on protein concentration. We observe an unexpected nonmonotonic trend in the measured terahertz absorbance of the five helix bundle protein * 6 -85 as a function of the protein: water molar ratio. The trend can be explained by overlapping solvation layers around the proteins. Molecular dynamics simulations indicate water dynamics in the solvation layer around one protein to be distinct from bulk water out to Ϸ10 Å. At higher protein concentrations such that solvation layers overlap, the calculated absorption spectrum varies nonmonotonically, qualitatively consistent with the experimental observations. The experimental data suggest an influence on the correlated water network motion beyond 20 Å, greater than the pure structural correlation length usually observed.solvation dynamics ͉ THz spectroscopy ͉ lambda repressor ͉ molecular modeling W ater molecules interact with proteins on many length and time scales. Although the dynamics of the hydration water occurs on the picosecond time scale, ''slaving'' to fast solvent modes profoundly affects the slower but larger-scale protein motions (1). In return the protein influences the structure and dynamics of surrounding water molecules (2). X-ray crystallography has revealed ordered water structure around polar and charged sidechains (3), as well as cooperative insertion of water into hydrophobic cavities (4). Dielectric spectroscopy extends the time scale from microseconds down to 0.1 ns (5). Experiments have been extended to the THz range in films and crystals, probing motions on the picosecond time scale (6, 7). Hydrated protein powders probed by inelastic neutron scattering (0.1-100 ps) or solid-state NMR (nanoseconds) reveal that slower protein time scales and faster solvent time scales indeed show correlated dynamics (8). On the fastest time scales, 2D infrared spectroscopy and fluorescence of surface residues provide local probes of the dynamics in the femtosecond to picosecond range (9, 10). Coupling of modeling with experiments has revealed complex solvation structure around small biomolecules (11, 12), bridging our microscopic structural and thermodynamic understanding of biosolvation.Terahertz absorption spectroscopy of biomolecules fully solvated in water yields direct informa...

We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg∕ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.enzymatic activity | FRET | folding kinetics | thermal denaturation | protein conformational changes P hosphoglycerate kinase (PGK) is a 415-residue metabolic enzyme that produces ATP and is composed of two roughly equally sized subunits connected by a flexible hinge (1). In the crystal structure, the ADP and diphosphoglycerate binding sites, each located at an N and C subunit, are separated. It has been suggested that a large-scale conformational change (2) is necessary to bring the two subunits together when the phosphoryl group is catalytically transferred, and a hinge-bending mechanism has been postulated (3), bringing together both substrates at the inner surfaces of the C and N subdomains (4, 5).It is still unclear how the conformational and folding dynamics of PGK is affected by the interior of a cell, which is heavily crowded by macromolecules (6, 7). Various computational and theoretical studies have been developed to address the effect of volume exclusion exerted by surrounding macromolecules on protein activity inside cells, called the "macromolecular crowding effect" (8). This effect, in addition to weak chemical interactions between proteins and crowders (9), can stabilize the folded states of a protein relative to the unfolded state (10), perturb folding barriers (11,12), and alter folding rates (13) and folding routes (14).Macromolecular crowding could selectively stabilize one folded protein structure over another (8,(15)(16)(17), particularly for proteins that are structurally malleable so their domains aligned in different orientations would have similar free energies (18). Thus, what we regard as the native structur...

Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23-49 degrees C showed that the cell environment influences protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.

The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.

We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.

Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: (i) short-range direct interaction of the protein surface with the growing ice face and (ii) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein-water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis. We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate.hydration dynamics | THz spetroscopy A ntifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are classes of proteins that suppress ice growth in organisms and thereby enable their survival in subfreezing habitats (1). Despite their similar function, many distinct structures have been identified so far. AFPs have been identified in several organisms, including polar fish (2), insects (3), bacteria (4), and plants (5). Their common characteristic is the depression of the freezing temperatures of ice growth of a solution without depressing the melting point equilibrium of protein solutions. This nonequilibrium phenomenon leads to a difference between the freezing and melting temperature, which is referred to as thermal hysteresis (TH). TH is used as a characteristic measure for antifreeze activity of an AFP (6). AFGPs and AFPs, as extracted from the blood of polar fish, usually exhibit up to 2°of TH activity and are termed moderately active AFPs, whereas insect AFPs can exhibit over 5°of TH and therefore, are referred to as hyperactive AFPs. The work by Raymond and DeVries (7,8) proposed a mechanism in which freezing point depression is achieved by an adsorption-inhibition mechanism, in which the proteins recognize and bind "quasiirreversibly" to an ice surface, thereby preventing growth of ice crystals. The adsorption of the protein is thought to prevent macroscopic ice growth in the hysteresis gap, but microscopic growth occurs at the interface in the form of highly curved fronts between adsorbed antifreeze molecules. This effect will cause a decrease of the local freezing temperature because of the Kelvin effect, while leaving the melting temperature relatively unaffected (7). As recently pointed out in the work by Sharp (9), antifreeze activity involves one of the most difficult recognition problems in biology, the distinction between water as liquid and ice. The initially proposed mechanism builds on a local mechanism. In particular, threonine (Thr) residues were proposed to play a decisive role: their hydroxyl groups were thought to be responsible for the high af...

The role of water in the functioning of proteins has been a hot topic over the years. We use terahertz (THz) spectroscopy as an experimental tool to probe the protein-induced fast solvation dynamics of ubiquitin. In order to investigate the effect of protein flexibility on the changes in the solvation dynamics, we have measured the concentration-dependent THz absorption of several site-specific ubiquitin mutants. The observed non-linear dependence of absorption on concentration is a signature of a long-range hydration shell with properties distinct from bulk water. We determined a dynamical hydration shell of a thickness of at least 18 A on the protein surface. This exceeds the static hydration layer as it is typically observed by scattering methods (3 A) by far. We also conclude that any increase in flexibility obtained by side-chain truncations that decrease the structural rigidity of the protein results in more bulk-like behaviour of the dynamical hydration shell. Furthermore, our THz measurements show that a single phenylalanine-to-tryptophan substitution to introduce a fluorescent marker leads to measurable changes in the solvation dynamics.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) enable the survival of organisms living in subfreezing habitats and serve as preservatives. Although their function is known, the underlying molecular mechanism was not understood. Mutagenesis experiments questioned the previous assumption of hydrogen bonding as the dominant mechanism. We use terahertz spectroscopy to show that antifreeze activity is directly correlated with long-range collective hydration dynamics. Our results provide evidence for a new model of how AFGPs prevent water from freezing. We suggest that antifreeze activity may be induced because the AFGP perturbs the aqueous solvent over long distances. Retarded water dynamics in the large hydration shell does not favor freezing. The complexation of the carbohydrate cis-hydroxyl groups by borate suppresses the long-range hydration shell detected by terahertz absorption. The hydration dynamics shift toward bulk water behavior strongly reduces the AFGP antifreeze activity, further supporting our model.