Structural effects of hydration: studies of lysozyme by 13C solids NMR

13C-nmr spectra of lysozyme obtained at 50.3 MHz using both static and magic-angle-spinning-cross-polarization methods are reported at several water contents. The line widths and consequent resolution in the hydrated material is substantially improved over that in the lyophilized protein. The line narrowing is not commensurate with loss of a proton-carbon dipole-dipole coupling or dramatic changes in the relaxation parameters characterizing magnetization transfer from protons to carbon in the Hartmann-Hahn cross-polarization experiment. We interpret these data in terms of the water inducing a decrease in the distribution of local conformations sampled by the protein, although the magnitude of the conformational reorientations required to account for the data are not necessarily large nor do they imply a major unfolding of the protein on dehydration.     

Active-site ionizations of papain. An evaluation of the potentiometric difference titration method

The underlying assumption of the potentiometric difference titration method, as applied to the evaluation of the sulfhydryl-dependent ionizations in the active site of papain, is that the pKa of His-159 is independent of whether the neighboring sulfhydryl (Cys-25) is protonated or methylthiolated. That this idealized assumption may not strictly apply is indirectly indicated by the larger pKa of His-159 in S-methylpapain versus that in S-methylthiopapain, as determined from fluorometric titrations (delta pKa = 0.32 +/- 0.05, 25 degrees C). On the basis of the Wegscheider principle of the equivalence of protons and methyl groups, the potentiometric difference titration method will underestimate the concentration of thiolate-imidazolium ion pair in the active site versus that of the thiol-imidazole tautomer, provided that there is no significant H-bonding interaction in the latter species.     

A systematic method for analysing the protein hydration structure of T4 lysozyme

A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water–protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters. Copyright © 2013 John Wiley & Sons, Ltd.     

Cavity hydration as a gateway to unfolding: an NMR study of hen lysozyme at high pressure and low temperature

We have used low temperatures (down to − 20 °C) and high pressures (up to 2000 bar) to populate low-lying excited state conformers of hen lysozyme, and have analyzed their structures site-specifically using ¹⁵N/¹H two-dimensional HSQC NMR spectroscopy. The resonances of a number of residues were found to be selectively broadened, as the temperature was lowered at a pressure of 2000 bar. The resulting disappearance of cross-peaks includes those of residues in the β-domain of the protein and the cleft between the β- and α-domains, both located close to water-containing cavities. The results indicate that low-lying excited state conformers of hen lysozyme are characterized by slowly fluctuating local conformations around these cavities, attributed to the opportunities for water molecules to penetrate into the cavities. Furthermore, we have found that these water-containing cavities are conserved in similar positions in lysozymes from a range of different biological species, indicating that they are a common evolutionary feature of this family of enzymes.     

Dynamics of lysozyme and its hydration water under an electric field

The effects of a static electric field on the dynamics of lysozyme and its hydration water are investigated by means of incoherent quasi-elastic neutron scattering (QENS). Measurements were performed on lysozyme samples, hydrated respectively with heavy water (D ₂O) to capture the protein dynamics and with light water (H ₂O), to probe the dynamics of the hydration shell, in the temperature range from 210 < T < 260 K. The hydration fraction in both cases was about ～ 0.38 gram of water per gram of dry protein. The field strengths investigated were respectively 0 kV/mm and 2 kV/mm ( ～2 × 10 ⁶ V/m) for the protein hydrated with D ₂O and 0 kV and 1 kV/mm for the H ₂O-hydrated counterpart. While the overall internal protons dynamics of the protein appears to be unaffected by the application of an electric field up to 2 kV/mm, likely due to the stronger intra-molecular interactions, there is also no appreciable quantitative enhancement of the diffusive dynamics of the hydration water, as would be anticipated based on our recent observations in water confined in silica pores under field values of 2.5 kV/mm. This may be due to the difference in surface interactions between water and the two adsorption hosts (silica and protein), or to the existence of a critical threshold field value E _c ～2–3 kV/mm for increased molecular diffusion, for which electrical breakdown is a limitation for our sample.     

Influence of hydration on the dynamics of lysozyme

Quasielastic neutron and light-scattering techniques along with molecular dynamics simulations were employed to study the influence of hydration on the internal dynamics of lysozyme. We identified three major relaxation processes that contribute to the observed dynamics in the picosecond to nanosecond time range: 1), fluctuations of methyl groups; 2), fast picosecond relaxation; and 3), a slow relaxation process. A low-temperature onset of anharmonicity at T approximately 100 K is ascribed to methyl-group dynamics that is not sensitive to hydration level. The increase of hydration level seems to first increase the fast relaxation process and then activate the slow relaxation process at h approximately 0.2. The quasielastic scattering intensity associated with the slow process increases sharply with an increase of hydration to above h approximately 0.2. Activation of the slow process is responsible for the dynamical transition at T approximately 200 K. The dependence of the slow process on hydration correlates with the hydration dependence of the enzymatic activity of lysozyme, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lysozyme.     

HEW lysozyme salting by high-concentration NaCl solutions followed by titration calorimetry

Concentration dependence of NaCl salting of 0-1.5 mM lysozyme solution in 0.1 M sodium acetate buffer, pH 4.25, was investigated for NaCl concentration varying up to 0.9 M. Calorimetric experiments demonstrated that depending on the salt concentration the estimated number of the binding sites on the lysozyme surface varied in the range of 5 up to 13, and the increase of salt concentration caused the decrease of the number of accessible sites. The small, but significant, local maximum centered at 0.63 M NaCl concentration indicated the specific salting-out of the lysozyme accompanied by binding of approximately 2-3 chloride anions. Generalized McMillan and Mayer's approach reduced to the third-order virial coefficients demonstrates the domination of lysozyme aggregation upon salt addition (a(21)-h(xxy)) and salt organization on the lysozyme surface (a(12)-h(xyy)) processes.     

An active-site titration method for lipases

A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.     

Structural characteristics of hydration sites in lysozyme

A new method is presented for determining the hydration site of proteins, where the effect of structural fluctuations in both protein and hydration water is explicitly considered by using molecular dynamics simulation (MDS). The whole hydration sites (HS) of lysozyme are composed of 195 single HSs and 38 clustered ones (CHS), and divided into 231 external HSs (EHS) and 2 internal ones (IHS). The largest CHSs, ‘Hg’ and ‘Lβ’, are the IHSs having 2.54 and 1.35 mean internal hydration waters respectively. The largest EHS, ‘Clft’, is located in the cleft region. The real hydration structure of a CHS is an ensemble of multiple structures. The transition between two structures occurs through recombinations of some H-bonds. The number of the experimental X-ray crystal waters is nearly the same as that of the estimated MDS hydration waters for 70% of the HSs, but significantly different for the rest of HSs.     

Multinuclear NMR study of enzyme hydration in an organic solvent

Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer's physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by 17O- and 2H-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state 2H-NMR spectra of enzyme-bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.     

Dynamics of hydration in hen egg white lysozyme

We investigate the hydration dynamics of a small globular protein, hen egg-white lysozyme. Extensive simulations (two trajectories of 9 ns each) were carried out to identify the time-scales and mechanism of water attachment to this protein. The location of the surface and integral water molecules in lysozyme was also investigated. Three peculiar temporal scales of the hydration dynamics can be discerned: two among these, with sub-nanosecond mean residence time, tau(w), are characteristic of surface hydration water; the slower time-scale (tau(w) approximately 2/3 ns) is associated with buried water molecules in hydrophilic pores and in superficial clefts. The computed tau(w) values in the two independent runs fall in a similar range and are consistent with each other, thus adding extra weight to our result. The tau(w) of surface water obtained from the two independent trajectories is 20 and 24 ps. In both simulations only three water molecules are bound to lysozyme for the entire length of the trajectories, in agreement with nuclear magnetic relaxation dispersion estimates. Locations other than those identified in the protein crystal are found to be possible for these long-residing water molecules. The dynamics of the hydration water molecules observed in our simulations implies that each water molecule visits a multitude of residues during the lifetime of its bound with the protein. The number of residues seen by a single water molecule increases with the time-scale of its residence time and, on average, is equal to one only for the water molecules with shorter residence time. Thus, tau(w) values obtained from inelastic neutron scattering and based on jump-diffusion models are likely not to account for the contribution of water molecules with longer residence time.     

The influence of hydration on the conformation of lysozyme studied by solid-state 13C-nmr spectroscopy

¹³C proton decoupled cross-polarization magic-angle spinning nmr spectra of lysozyme are reported as a function of hydration. Increases in hydration level enhance the resolution of the spectra, particularly in the aliphatic region, but has no significant effect on either the rotating frame proton spin–lattice relaxation time or the cross-relaxation time. The enhancement in spectral resolution with hydration is attributed to a decrease in the distribution of isotropic chemical shifts, which reflects a decrease in the distribution of conformational states sampled by the protein. Changes in the distribution of isotropic chemical shifts occur after the addition of water to the charged groups as coverage of the polar side chains and peptide groups takes place. The onset of this behavior occurs at a hydration level of about, 0.1–0.2 g water/g protein and is largely complete at about 0.3 g water/g protein, the same hydration range where changes in the heat capacity are observed. That hydrogen exchange of buried protons can occur at hydration levels significantly lower than those at which changes in the distribution of conformational states are first observed suggests that some motions that mediate exchange are already present in the dry protein. The preservation of efficient dipolar coupling indicates that the conformational rearrangements that do-occur on hydration are small and do not involve any significant overall expansion of free volume or weakening of interactions that would increase the reorientational freedom of protein groups. © 1993 John Wiley & Sons, Inc.