Study of the dynamics of human serum albumin by coherent Rayleigh dispersion of Mossbauer radiation]

The measurements of angle dependencies of total and elastic Rayleigh scattering of Mossbauer radiation intensities have been performed for human serum albumin (HSA) with hydration degrees h = 0.13 and h = 0.4. The extended model was developed for calculating the inelastic intensity of Rayleigh scattering. Original data for HSA and published data on met-Mb were fitted within the frame of this model. The best agreement with experiment was obtained when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative (mechanical) motions of segments (most probable alpha-helices). Long-range correlated motions are essential at low hydration degree. The possibilities of application of the coherent version of RSMS technique are described.     

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state.

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10)s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8)s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8)s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10)s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.     

Hydration dynamics near a model protein surface

The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering (QENS) and molecular dynamics (MD) simulation for both the completely deuterated and completely hydrogenated leucine monomer. The NALMA-water system and the QENS data together provide a unique study for characterizing the dynamics of different hydration layers near a prototypical hydrophobic side chain and the backbone of which it is attached. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational and rotational water dynamics at the highest solute concentrations are found to be highly suppressed as characterized by long residential time and slow diffusion coefficients. The analysis of the more dilute concentration solutions models the first hydration shell with the 2.0 M spectra. We find that for outer layer hydration dynamics that the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis of the first hydration shell water dynamics shows spatially heterogeneous water dynamics, with fast water motions near the hydrophobic side chain, and much slower water motions near the hydrophilic backbone. We discuss the hydration dynamics results of this model protein system in the context of protein function and protein-protein recognition.     

Internal motions of actin characterized by quasielastic neutron scattering

Quasielastic neutron scattering (QENS) experiments were carried out on powders of F-actin and G-actin hydrated with D₂O to characterize the internal dynamics on the picosecond time scale and the Ångstrom length scale. To investigate the effects of hydration, the measurements were done on samples at hydration ratio (h) of 0.4 (mg D₂O/mg protein), containing only the first layer of hydration water, and at h = 1.0, containing more layers of water. The QENS spectra, obtained from the measurements at two energy resolutions of 110 and 15 μeV, indicated that the internal motions of both F-actin and G-actin have distributions of motions with distinct correlation times and amplitudes. Increasing hydration changes relative populations of these distinct motions. The effects of hydration were shown to be different between F-actin and G-actin. Elastic incoherent neutron scattering measurements provided the concerted results. The observed effects were interpreted in terms of the dynamical heterogeneity of the actin molecule: in G-actin, more surface loops become flexible and undergo diffusive motions of large amplitudes, whereas in F-actin the molecular interactions that keep the polymerized state suppress the large motions of the surface loops involved with polymerization so that the population of atoms undergoing large motions can increase only to a lesser degree.     

Osmolyte trimethylamine-N-oxide does not affect the strength of hydrophobic interactions: origin of osmolyte compatibility

Osmolytes are small organic solutes accumulated at high concentrations by cells/tissues in response to osmotic stress. Osmolytes increase thermodynamic stability of folded proteins and provide protection against denaturing stresses. The mechanism of osmolyte compatibility and osmolyte-induced stability has, therefore, attracted considerable attention in recent years. However, to our knowledge, no quantitative study of osmolyte effects on the strength of hydrophobic interactions has been reported. Here, we present a detailed molecular dynamics simulation study of the effect of the osmolyte trimethylamine-N-oxide (TMAO) on hydrophobic phenomena at molecular and nanoscopic length scales. Specifically, we investigate the effects of TMAO on the thermodynamics of hydrophobic hydration and interactions of small solutes as well as on the folding-unfolding conformational equilibrium of a hydrophobic polymer in water. The major conclusion of our study is that TMAO has almost no effect either on the thermodynamics of hydration of small nonpolar solutes or on the hydrophobic interactions at the pair and many-body level. We propose that this neutrality of TMAO toward hydrophobic interactions-one of the primary driving forces in protein folding-is at least partially responsible for making TMAO a "compatible" osmolyte. That is, TMAO can be tolerated at high concentrations in organisms without affecting nonspecific hydrophobic effects. Our study implies that protein stabilization by TMAO occurs through other mechanisms, such as unfavorable water-mediated interaction of TMAO with the protein backbone, as suggested by recent experimental studies. We complement the above calculations with analysis of TMAO hydration and changes in water structure in the presence of TMAO molecules. TMAO is an amphiphilic molecule containing both hydrophobic and hydrophilic parts. The precise balance of the effects of hydrophobic and hydrophilic segments of the molecule appears to explain the virtual noneffect of TMAO on the strength of hydrophobic interactions.     

Function of the F1-motor (F1-ATPase) of ATP synthase by apolar-polar repulsion through internal interfacial water

THESIS: Within the structurally-confined internal aqueous cavity of the F1-motor of ATP synthase, function results from free energy changes that shift the balance between interfacial charge hydration and interfacial hydrophobic hydration. TRANSITION STATE DESCRIPTION: At the beta-P end of ADP x Mg occurs an inorganic phosphate, P(i). This P(i) resides at the base of a water-filled cleft that functions like an aperture to focus, into an aqueous chamber, a competition for hydration (an apolar-polar repulsion) between charged phosphate and hydrophobic surface of the gamma-rotor. Two means available for the phosphate and the hydrophobic surface to improve their hydration free energies are physically to separate by rotation of the gamma-rotor or chemically to combine P(i) with ADP to form less charged ATP. This proposal derives from calculated changes in Gibbs free energy for hydrophobic association of amino acid side chains and chemical modifications thereof and from experimentally demonstrated water-mediated repulsion between hydrophobic and charged sites that resulted from extensive studies on designed elastic-contractile model proteins.     

Dynamics of hydration water in deuterated purple membranes explored by neutron scattering

The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H(2)O and D(2)O, respectively, revealed that membrane and water motions on the ns-ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049-18054, 2007). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H(2)O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D(2)O (Weik et al. in J Mol Biol 275:632-634, 2005), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins.     

Hydration dependence of active core fluctuations in bacteriorhodopsin

We used neutron scattering and specific hydrogen-deuterium labeling to investigate the thermal dynamics of isotope-labeled amino acids and retinal, predominantly in the active core and extracellular moiety of bacteriorhodopsin (BR) in the purple membrane and the dynamical response to hydration. Measurements on two neutron spectrometers allowed two populations of motions to be characterized. The lower amplitude motions were found to be the same for both the labeled amino acids and retinal of BR and the global membrane. The larger amplitude dynamics of the labeled part, however, were found to be more resilient than the average membrane, suggesting their functional importance. The response to hydration was characterized, showing that the labeled part of BR is not shielded from hydration effects. The results suggest that the inhibition of high-amplitude motions by lowering hydration may play a key role in the slowing down of the photocycle and the proton pumping activity of BR.     

Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP

We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<∼200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <∼180–190 K and is not affected by the proteins’ methyl group rotations. The dynamics of the hydration water exhibits changes at ∼180–190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.     

Equilibrium fluctuations in myoglobin and lysozyme

The angular dependencies of inelastic intensities of Rayleigh scattering of Moessbauer radiation were measured for myoglobin and lysozyme (in the hydration range h = 0.05-0.7). The data were fitted within the framework of model, when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative motions of segments. The best agreement with the experiment at h > 0.05 was obtained when individual motions of small groups together with the cooperative motions of alpha-helices and beta-sheets for lysozyme, and alpha-helices for myoglobin were considered. At further hydration (h = 0.45), mean-square displacements (x2) of both types of motions strongly increase with the increase in hydration degree, while the motions with a large correlation radius (not less than macromolecule radius) remain nearly the same as for h = 0.05. The results of the study of the radial distribution function deduced by Fourier-transform from the diffuse x-ray measurements together with RSMR data allow one to conclude that the water during protein hydration competes with the intramolecular hydrogen bonds, loosens the protein and increases the internal dynamics. Concurrently, water arranges the ordering of macromolecule, which takes the native structure at h = 0.4-0.7. The analysis of auto and cross-correlation functions of bending fluctuations of alpha-helices in the large domain of lysozyme performed by molecular dynamics allows one to come to the final conclusion that it is the difference in the structural organization of myoglobin and lysozyme and not the presence of SS-bonds in lysozyme macromolecule that is responsible for different structural fluctuations in these proteins.     

Hydration affects both harmonic and anharmonic nature of protein dynamics

To understand the effect of hydration on protein dynamics, inelastic neutron-scattering experiments were performed on staphylococcal nuclease samples at differing hydration levels: dehydrated, partially hydrated, and hydrated. At cryogenic temperatures, hydration affected the collective motions with energies lower than 5 meV, whereas the high-energy localized motions were independent of hydration. The prominent change was a shift of boson peak toward higher energy by hydration, suggesting a hardening of harmonic potential at local minima on the energy landscape. The 240 K transition was observed only for the hydrated protein. Significant quasielastic scattering at 300 K was observed only for the hydrated sample, indicating that the origin of the transition is the motion activated by hydration water. The neutron-scattering profile of the partially hydrated sample was quite similar to that of the hydrated sample at 100 K and 200 K, whereas it was close to the dehydrated sample at 300 K, indicating that partial hydration is sufficient to affect the harmonic nature of protein dynamics, and that there is a threshold hydration level to activate anharmonic motions. Thus, hydration water controls both harmonic and anharmonic protein dynamics by differing means.     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

Dynamic structure of subtilisin-eglin c complex studied by normal mode analysis

Normal mode analysis of subtilisin-eglin c complex was performed to investigate the dynamics at the interface between the enzyme and the inhibitor. The internal motions of the complex calculated from the normal modes were divided into three parts: the internal motions changing the shape of each molecule, the external rigid-body motions changing their mutual dispositions, and the coupling between the internal and external motions. From the results of the analysis, the following characteristic features were found in the dynamics at the interface regions: 1) negative correlation between the internal and external motions within each molecule, and 2) positive correlation between the external motions of the two molecules. The former decreases the apparent amplitudes of motions at the interface. The latter minimizes the interference between individual motions of the two molecules. These dynamic characteristics allow the enzyme and the inhibitor to move as freely as possible. This finding suggests that the experimental evidence of the large entropy gain on binding should be attributed not only to strong hydrophobic interactions, but also to the dynamic structure of the complex, which is found to minimize an unavoidable loss of the conformational entropy on binding. Proteins 32:324–333, 1998. © 1998 Wiley-Liss, Inc.     

Hydration of a hydrophobic cavity and its functional role: a simulation study of human interleukin-1beta

Molecular dynamics simulations reveal that the hydrophobic cavity in human cytokine Interleukin-1beta is hydrated and can dynamically accommodate between one and four water molecules. These waters have residence times > 500 ps and can give rise to detectable NOEs, in agreement with NMR observations of Ernst et al. (Science 1995; 267:1813-1817). The waters also display high positional disorder within the cavity, which explains why they have not been resolved crystallographically. The average distribution of water molecules over time within the cavity matches well the low resolution electron density extracted by Yu et al. (Proc Natl Acad Sci 1999; 96:103-108). The water molecules hydrate the hydrophobic cavity preferentially as complex clusters. These clusters result from a combination of hydrogen bonds between the waters and stabilizing interactions between the waters and aromatic rings forming the cavity. Free energy estimates suggest that it takes 4-waters to hydrate the cavity in a thermodynamically stable manner leading to a gain in free energy of transfer from bulk of approximately approximately 3.6 kcal/mol. This arises from the existence of the water clusters in multiple hydrogen bonded states. In addition, the waters are found to migrate either individually or as clusters out of the cavity through several pathways. The upper limit for one-dimensional diffusion of the waters within the protein matrix is 4 A/ps (relative to 6 A/ps for bulk). Simulations reveal pathways in addition to those identified crystallographically, with motions controlled by the rotations of sidechains. We find that only when the hydrophobic cavity is hydrated, do correlated motions couple distant sites with the sites that make contact with the receptor and this data partly offers an explanation of experimental mutagenesis data. Simulations, together with recent observations based on mutagenesis by Heidary et al. (J Mol Biol 2005; 353:1187-1198) that hydrogen bond networks couple motions across long distances in interleukin-1beta, lead us to hypothesize that the hydration of the cavity (conserved across mammals) can thermodynamically enhance hydrogen bond networks to enable coupling across long distances by acting as a plug and this in turn enables a kinetic control of the rate of transmission of signals.     

Fundamental measure theory of hydrated hydrocarbons

To calculate the solvation of hydrophobic solutes, we have developed a method based on the fundamental measure treatment of density functional theory. This method allows us to carry out calculations of density profiles and the solvation energy for various hydrophobic molecules with high accuracy. We have applied the method to the hydration of various hydrocarbons (linear, branched and cyclic). The calculations of the entropic and enthalpic parts are also carried out. We have examined the question of the temperature dependence of the entropy convergence. Finally, we have calculated the mean force potential between two large hydrophobic nanoparticles immersed in water. Proceedings of “Modeling Interactions in Biomolecules II”, Prague, September 5th–9th, 2005.     

Evidence of protein collective motions on the picosecond timescale

We investigate the presence of structural collective motions on a picosecond timescale for the heme protein, cytochrome c, as a function of oxidation and hydration, using terahertz (THz) time domain spectroscopy and molecular dynamics simulations. The THz response dramatically increases with oxidation, with the largest increase for lowest hydrations, and highest frequencies. For both oxidation states the THz response rapidly increases with hydration saturating above ∼25% (g H₂O/g protein). Quasiharmonic vibrational modes and dipole-dipole correlation functions were calculated from molecular dynamics trajectories. The collective mode density of states alone reproduces the measured hydration dependence, providing strong evidence of the existence of these motions. The large oxidation dependence is reproduced only by the dipole-dipole correlation function, indicating the contrast arises from diffusive motions consistent with structural changes occurring in the vicinity of buried internal water molecules. This source for the observed oxidation dependence is consistent with the lack of an oxidation dependence in nuclear resonant vibrational spectroscopy measurements.     

The kinetics of interactions of bilirubin with lipid bilayers and with serum albumin.

Rate constants for the hydration of bilirubin bound to unilamellar bilayers of dioleoylphosphatidylcholine and albumin were measured by stopped-flow methods. Rate constants for association of bilirubin with these vesicles and albumin were calculated from measured rate constants for dissociation and the equilibrium binding constants of bilirubin and lipids or albumin. Rate constants for hydration (dissociation) for bilirubin bound to dioleoylphosphatidylcholine and albumin were 71 s-1 and 1.8 s-1 respectively. Rate constants for association were 4.0 10(7) s-1 and 1.1 10(9) M-1 s-1, respectively. Both rates for interactions of bilirubin with bilayers were essentially independent of temperature in the range 0-40 degrees C, indicating that barriers to entry and exit of bilirubin from bilayers were entropic. Rates of transbilayer movement of bilirubin in dioleoylphosphatidylcholine were too fast to resolve by measuring rates of hydration of bilirubin. Rate constants for hydration of bilirubin bound to bilayers with less avidity for bilirubin as compared with dioleoylphosphatidylcholine also were too fast to measure with stopped-flow methods. In addition to providing details of the energetic basis for interactions between bilirubin and membranes, the data allow for calculating the maximal rates at which bilirubin could transfer spontaneously from sites on albumin in blood to the interior of cells. The data show, in this regard, that this rate is 10-50 fold faster than measured rates of uptake of bilirubin by intact liver.     

Dynamics of heme complexed with human serum albumin: a theoretical approach

Human serum albumin (HSA) is the most abundant protein in the blood serum. It binds several ligands and has an especially strong affinity for heme, hence becoming a natural candidate for oxygen transport. In order to analyze the interaction of HSA-heme, molecular dynamics simulations of HSA with bound heme were performed. Based on the results of X-ray diffraction, the binding site of the heme, localized in subdomain IB, was considered. We analyzed the fluctuations and their correlations along trajectories to detect collective motions. The role of H bonds and salt bridges in the stabilization of heme in its pocket was also investigated. Complementarily, the localization of water molecules in the hydrophobic pocket and the interaction with heme were discussed.     

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.     

NMR characterizations of the ice binding surface of an antifreeze protein

Antifreeze protein (AFP) has a unique function of reducing solution freezing temperature to protect organisms from ice damage. However, its functional mechanism is not well understood. An intriguing question concerning AFP function is how the high selectivity for ice ligand is achieved in the presence of free water of much higher concentration which likely imposes a large kinetic barrier for protein-ice recognition. In this study, we explore this question by investigating the property of the ice binding surface of an antifreeze protein using NMR spectroscopy. An investigation of the temperature gradient of amide proton chemical shift and its correlation with chemical shift deviation from random coil was performed for CfAFP-501, a hyperactive insect AFP. A good correlation between the two parameters was observed for one of the two Thr rows on the ice binding surface. A significant temperature-dependent protein-solvent interaction is found to be the most probable origin for this correlation, which is consistent with a scenario of hydrophobic hydration on the ice binding surface. In accordance with this finding, rotational correlation time analyses combined with relaxation dispersion measurements reveals a weak dimer formation through ice binding surface at room temperature and a population shift of dimer to monomer at low temperature, suggesting hydrophobic effect involved in dimer formation and hence hydrophobic hydration on the ice binding surface of the protein. Our finding of hydrophobic hydration on the ice binding surface provides a test for existing simulation studies. The occurrence of hydrophobic hydration on the ice binding surface is likely unnecessary for enhancing protein-ice binding affinity which is achieved by a tight H-bonding network. Subsequently, we speculate that the hydrophobic hydration occurring on the ice binding surface plays a role in facilitating protein-ice recognition by lowering the kinetic barrier as suggested by some simulation studies.