A Study of Water–Protein Interactions by High-Resolution NMR Spectroscopy

The interaction between carbonic anhydrase B in the molten globule state and water molecules was studied by high-resolution NMR spectroscopy. NMR spin diffusion experiments revealed spin diffusion propagation from the protein to waters. This is a process of complex bioexponential kinetics presented in spin diffusion spectra as a change in water signal intensity dependent on the protein postexcitation time. Its reverse, spin diffusion propagation from waters to the protein, was also found. These phenomena are protein concentration- and temperature-dependent and shown to be possibly explained with the assumption that there exist water–protein complexes provoking formation of large branched associations. At a temperature above 309 K, a stepwise increase in the interaction between water and proteins occurs in these complexes. The formation of water–protein associations is induced by increasing temperature and/or protein concentration. In these associations, at normal temperature, the protein mobility is close to that of carbonic anhydrase B dimers.     

A study of interactions of carbonic anhydrase B with water and urea. I. Spin-diffusion parameters that determine individual and cooperative properties of molecules

The rigidity parameter (G), which is characteristic of protein compactness, was studied in native globular carbonic anhydrase B. The dependence of parameter G on power and excitation time of spin-diffusion was expressed analytically. We found out that native carbonic anhydrase B is able to form water-protein units that are probabilistically distributed with respect to their sizes. Large water-protein units can be detected by analyzing the spin-diffusion spectra. The excitation frequencies of spin-diffusion spectra were shifted far away from typical 1H NMR spectra of carbonic anhydrase B.     

Carbonic Anhydrase B Interactions with Water and Urea

High-resolution NMR spectroscopy has been used to study native carbonic anhydrase B unfolding with urea at pH 5.75 and T = 298 K. The rigidity parameter reflecting the effectiveness of spin diffusion (SD) displays a sigma-like dependence on urea concentration, which is characteristic of denaturing processes. The ratio between the integral intensities of urea and protein signals measured in SD spectra and normal 1D spectra are the same. This suggests the absence of a predominant interaction between urea and protein molecules. The concentration of large protein-solvent complexes rapidly increases at urea concentrations of 4.2–6.2 M, which is apparently related to the transition of the protein into the molten globule state. If the urea concentration is increased to 6.6 M, these complexes dissociate, and the polypeptide chain of carbonic anhydrase B becomes completely unfolded.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 497–503.Original Russian Text Copyright © 2005 by Prokhorov, Kutyshenko, Khristoforov.     

Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein

The peculiarities of thermal denaturation and interaction with water of the cycle-3 mutant of green fluorescent protein (GFP) were analyzed by NMR techniques and compared with those of bovine carbonic anhydrase II (BCA-II). Irreversible thermal denaturation was accompanied by massive GFP aggregation with no detectable accumulation of soluble denatured protein. Analysis of the spin diffusion data suggested that the internal part of the GFP β-can is involved in intensive interactions with water molecules. As a result, at high temperatures, the GFP structure does not unfold but rather breaks, consequently leading to enhanced protein aggregation. This is very different from typical BCA-II behavior.     

Studies of interactions of carbonic anhydrase B with water and urea: II. Spin diffusion of associates of protein intermediate state at 4.2 M of urea

The formation of carbonic anhydrase B associates (pH 5.7, urea concentration 4.2 M, 297 K) was studied as a function of protein concentration and time by nuclear magnetic resonance spectroscopy (spin diffusion method). It was found that the association process proceeds in two steps. The first step is relatively fast and cannot be controlled by our methods. During this step, persistent units are built. These consist of protein molecules that are able to interact with solvent molecules and with each other when protein solution contains 4.2 M of urea. Persistent units are relatively small (two, three protein molecules), and their mobility matches one of a single protein. The second step is slower, and throughout this step large structures are formed from persistent units. The parameters G* and S*, which characterize spin diffusion in a protein and a solvent, respectively (when spin diffusion excitation happens away from NMR spectral signals) are related to the probable size distribution of protein-solvent associates and are determined by their collective properties.     

Guanidine hydrochloride mediated unfolding of a carbonic anhydrase molten globule

Guanidine hydrochloride-induced unfolding of a carbonic anhydrase molten globule was studied by high-resolution NMR spectroscopy. The study resulted in estimation of the number of water and denaturant molecules bound to the molten globule at various denaturant concentrations in solution. When compared with the data on unfolding of native carbonic anhydrase, these estimates indicate that the unfolding is underlain by an increased local concentration of the denaturant near the protein molecule, which results from the increased ratio between guanidine hydrochloride-bound and protein-bound waters.     

A study of interaction of carbonic anhydrase B with water and urea: II. Spin diffusion of associates of protein intermediate state at 4.2 M urea

Formation of the associates of carbonic anhydrase B (pH 5.7, 4.2 M urea, and T = 297 K) as a function of protein concentration and time clapsed after preparation of solutions was studied by nuclear magnetic resonance spectroscopy (spin diffusion method). It was demonstrated that the association was a two-stage process. The initial (fast) stage, involving the formation of persistent blocks, was independent of the time elapsed after the solution preparation. A urea concentration of 4.2 M allows the protein molecules to interact with one another to form rather small persistent blocks in combination with solvent molecules, so that the mobility of each molecule remains nearly unchanged. The final (slow) stage is time-dependent and involves the formation of large structures from the persistent blocks. It is shown that parameters G* and S*, which characterize spin diffusion (in protein and solvent, respectively) when it is excited at frequencies remote from the NMR spectral signals, are related to the size probability distribution of the solvent-protein associates and are determined by their collective properties.     

Microcalorimetric study of the heat denaturation of carboanhydrase B

The microcalorimetric study of heat denaturation of carbonic anhydrase B has revealed that the process of denaturation of carbonic anhydrase B is accompanied by the formation of intermolecular complexes which are disrupted at a further increase of temperature. It is shown that zinc atoms stabilize the native state and do not influence the stability of intermolecular complexes.     

Guanidine Hydrochloride Unfolding of a Carbonic Anhydrase Molten Globule

Guanidine hydrochloride-induced unfolding of a carbonic anhydrase molten globule was studied by high-resolution nuclear magnetic resonance spectroscopy. The study resulted in estimation of the number of water and denaturant molecules bound to the molten globule at various denaturant concentrations in solution. When compared with the data on unfolding of native carbonic anhydrase, these estimates indicate that the unfolding is underlain by an increased local concentration of the denaturant near the protein molecule, which results from the increased ratio between guanidine hydrochloride-bound and protein-bound waters.     

Protein interaction with hydrated C60 fullerene in aqueous solutions

Physicochemical effects of hydrated C(60) fullerenes (HyFn) on serum albumin molecules were studied using ESR spin labeling and differential scanning microcalorimetry. Molecular-colloidal solution of hydrated C(60) fullerenes and their small spherical fractal clusters in water (C(60)FWS), was shown to stabilize protein hydration, and decrease specific surface energy in water-protein matrix in salt solutions. The mechanism of HyFn interaction with protein is discussed in terms of HyFn induced formation of protein clusters and phase transition of hydration water.     

The sequestration of hydroxyl ions by C2 in liquid water: useful physiological roles for a reversible complex formation in the presence of protein catalysts

A second function of carbonic anhydrase (CA) isoforms has already been proposed. This involves the dispersal of complexes in which six carbon dioxide molecules sequester a hydroxyl ion when the gas reacts with liquid water. The semi-catalytic reaction does not require the formation of bicarbonate as an essential corollary. This function is, therefore, a likely activity of carbonic anhydrase related proteins that have recently been discovered and which lack the active zinc site essential for the hydration of carbon dioxide. Re-examination of possible functions for the complex of six CO2 molecules with a hydroxyl anion have brought to light several circumstances where the presence of fully reversible complexes could have physiological advantages. A catalytic synthesis and dissolution of the complexes could thus be the important function for the carbonic anhydrase-related proteins (CA-RP) molecules as well as of some CA isoforms. The possible mechanisms for this extended second catalytic function and examples are briefly discussed.     

Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR

Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant Asn51 residue, seen in all known homeodomain/DNA structures, is critical for binding affinity and specificity.     

Water-protein interactions in the molten-globule state of carbonic anhydrase b: an NMR spin-diffusion study

We have used the homonuclear Overhauser effect (NOE) to characterize a model protein: carbonic anhydrase B. We have obtained NOE difference spectra for this protein, centering the on-resonance signals either at the methyl-proton or at the water-proton signals. The spin-diffusion spectra obtained as a function of protein concentration and temperature provide direct evidence of much greater protein-water interaction in the molten-globule state than in the native and denatured states. Furthermore, although the protein loses its gross tertiary structure in both the molten-globule and denatured states, it remains almost as compact in its molten-globule state as it is in the native state. The spin-diffusion spectra, obtained as a function of a variable delay time after the saturation pulse, allowed us to measure the relaxation times of several types of proton in the solution. These spectra contain enough information to distinguish between those water molecules solvating the protein and the free ones present as bulk water.     

Deuterium NMR of water in immobilized protein systems.

Deuterium NMR spectra are reported for lysozyme crystals, powders, and frozen solutions. At high water contents the spectrum is a superposition of a narrow central component and a quadrupole doublet. The quadrupole splitting and the relaxation rates of both components, monitored as a function of water content and temperature, are discussed in terms of models for the water-protein interaction. The anisotropy of the water molecule motion is clearly demonstrated by the deuterium quadrupole splitting observed in the protein single crystal, but such splittings were not found in protein powders and frozen protein solutions. We therefore suggest that the most useful view of such data is to consider the water-protein interactions at the surface to be mixed rapidly and that a distribution of interactions be invoked rather than an oversimplified view often taken of a two or n-site mixing where n is small.     

Carbonic anhydrase B interactions with water and urea

Carbonic anhydrase B unfolding with urea (pH 5.7, T = 298 K) was studied by high-resolution NMR spectroscopy. The effectiveness of spin-diffusion influencing compactness of the protein molecule can be described with the rigidity parameter G. Parameter G displays sigma-like characteristic behavior when concentration increases. The ratio between integral intensities of urea and protein signals in spin-diffusion and normal 1D spectra are the same. This suggests that there is no predominant urea-protein molecular interaction. The concentration of large protein-solvent associates increases rapidly at urea levels of 4.2-6.2 M implying that protein molecule shifts to a molten globule state. Protein-solvent associates are dissipating with urea concentration increase to above 6.6 M when carbonic anhydrase B polypeptide chain is completely unfolded.     

Role of arginine in the stabilization of proteins against aggregation

The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.     

Hydrophilicity of cavities in proteins

Water molecules inside cavities in proteins constitute integral parts of the structure. We have sought a quantitative measure of the hydrophilicity of the cavities by calculating energies and free energies of introducing a water molecule into these cavities. A threshold value of the water-protein interaction energy at −12 kcal/mol was found to be able to distinguish hydrated from empty cavities. It follows that buried waters have entropy comparable to that of liquid water or ice. A simple consistent picture of the energetics of the buried waters provided by this study enabled us to address the reliability of buried waters assigned in experiments.     

Zinc(II) complexes of tripodal peptides mimicking the zinc(II)-coordination structure of carbonic anhydrase.

Two new tripodal peptide ligands with histidine side chains have been synthesized and were shown to form stable zinc(II) complexes. Their NMR and mass spectra indicate a structure that is analogous to the active center of carbonic anhydrase. Both the ligands and the zinc complexes were titrated potentiometrically in order to obtain the pKa values for the coordinated water of the zinc complexes; due to the low solubility of the complexes only estimates could be obtained.     

Multy-state protein: Determination of carbonic anhydrase free-energy landscape

Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins—carbonic anhydrase B—is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations.     

Proton magnetic resonance studies of histidines in human, rhesus monkey, and bovine carbonic anhydrases.

Histidine C-2 proton resonances in rhesus monkey carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) and bovine carbonic anhydrase were investigated using 270-MHz proton magnetic resonance. The results suggest that there are extensive three-dimensional homologies between the human B and rhesus B enzymes and between the human C and bovine enzymes. Resonances from solvent exchangeable protons have been observed in the 11-16 ppm range in the NMR spectra of human carbonic anhydrases B and C and bovine carbonic anhydrase. Up to five of these are sensitive to changes of pH and the presence of inhibitors. Three of these resonances are assigned to NH protons of the metal coordinated imidazole groups. These results are discussed in relation to various models for the catalytic mechanism of carbonic anhydrase.