Helmut Pessen

Proton relaxation rates of water in dilute solutions of β-lactoglobulin determination of cross relaxation and correlation with structural changes by the use of two genetic variants of a self-associating globular protein

Abstract In order to relate resonance relaxation behavior to protein structural states, pulse Fourier transform NMR was employed to obtain water proton longitudinal and transverse relaxation rate ( R 1 and R 1 ϱ = R 2 ) of bovine β-lactoglobulins A and B in buffered solutions. Measurements at concentrations from 5 to 100 mg/ml were made at pH 4.65, 6.2 and 8.0, at 30 and 2°C, to monitor specific structural changes. The parameters characterizing the concentration dependence of the observed R 1 and R 2 were used to derive a number of quantities relating to protein-influenced water, including a hydration parameter h . Changes in h under the different sets of conditions were correlated with (a) the irreversible denaturation of this protein at pH 8.0, 2°C and (b) the dimer ⇄ octamer association at pH 4.65, 2°C. Corresponding correlation times, however, were low, indicating cross relaxation which had not manifested itself as nonexponential relaxation because of the large amount of water present. Differences in the extent of the ⇄ octamer association between genetic variants A and B allowed an evaluation of dynamics and extent of hydration from R 2 alone, assuming the absence of intermolecular interactions. Derived parameters were in agreement with hydrodynamic and X-ray values in the literature. Cross relaxation was likewise evaluated and was found to contribute to R 1 to a large extent. The results show that changes in proton relaxation rates in solutions of a globular protein occuring as genetic variants with different physical properties (such as β-lactoglobulin) can be utilized to detect variations in hydration corresponding to changes in molecular association and conformation, as well as to obtain cross relaxation and structural data.

[14] Measurements of protein hydration by various techniques

Publisher Summary This chapter deals with hydration measurements made on globular proteins in solution z by three techniques which have been found in recent years to afford certain useful insights: nuclear magnetic resonance (NMR) relaxation, small-angle X-ray scattering (SAXS), and hydrodynamics (velocity sedimentation). The latter two is treated together, because sedimentation, which cannot by itself give definitive values of hydration, is very useful in corroborating SAXS data. Conversely, sedimentation coefficients can be calculated independently from SAXS. NMR methodology should be treated in particular detail, both because of its potential utility for the present purpose of the chapter. Moreover, specific discussions of these methods are then followed by some general remarks, including an evaluation of the different approaches. It may be noted that correlations between SAXS and hydrodynamic methods other than sedimentation (viscosity, diffusion), and between various combinations of hydrodynamic data with each other, have been less successful.

Estimation of sedimentation coefficients of globular proteins: An application of small-angle X-ray scattering

A new semiempirical procedure is presented which relates solution small-angle X-ray scattering parameters to sedimentation coefficients. With this method, sedimentation coefficients were calculated for a set of 20 globular macromolecules with molecular weights ranging from 1.3 × 104 to 7.0 × 106; all were in excellent agreement with experimental values. Best results were arrived at by obtaining: (1) the Stokes radius in Svedbergs equation by way of the scattering volume V of the macromolecule instead of the commonly used partial specific volume v; and (2) the structural frictional ratio (ff0)s from an axial ratio derived from RGSV instead of the usual 3V(4πRG3 relationship, where RG is the radius of gyration and S is the external surface area of the molecule. This indicates that the frictional ratio is a function of the surface roughness of the macromolecule, in agreement with similar conclusions in the literature. In addition, structural parameters from the X-ray crystallographic structure are compared with those from small-angle X-ray scattering for a better insight into the contribution of hydration to the frictional coefficient.

Structure and mechanism of action of riboflavin-binding protein: Small-angle X-ray scattering, sedimentation, and circular dichroism studies on the holo- and apoproteins☆

Riboflavin-binding protein, a transport protein occurring in egg whites, binds riboflavin tightly at pH values above 4.5 but releases it readily at pH values below 4.0. Structural aspects of this biologically important binding were studied by several methods. Analysis of sedimentation equilibrium data gave an average molecular weight of 32,500 ± 1000 for all forms of the protein and showed the absence of changes in quaternary structure when riboflavin was bound at neutral pH or released at pH 3.7. Sedimentation velocity showed no change in tertiary structure on binding at pH 7.0 but revealed a significant change in sedimentation constant at pH 3.7. While circular dichroism showed no appreciable change in secondary structure, it gave evidence of a marked change in the aromatic region at the lower pH. Small-angle X-ray scattering, going from the holoprotein at neutral pH to the apoprotein at low pH, showed a small but significant increase in radius of gyration (19.8 ± 0.2 vs 20.6 ± 0.1 A) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 A3), surface (11,840 ± 120 vs 13,470 ± 140 A), and hydration (0.27 ± 0.01 vs 0.38 ± 0.01 g H2O/g dry protein). Hydration values were obtained from small-angle X-ray scattering in two different ways for comparison with those calculated from sedimentation coefficients by way of frictional coefficients (derived from two different dimensionless ratios based independently on the structural small-angle X-ray scattering data). For either form of the protein, the surface calculated from an ellipsoidal model could account for only about 62% of the surface found experimentally. The excess surface was ascribed to topographic features of the molecule. Relative changes in this new parameter, together with the circular dichroism data and the known association of riboflavin binding with aromatic residues, suggested the opening of an aromatic-rich cleft concomitant with the release of riboflavin as a consequence of lowered pH.

A deuteron and proton magnetic resonance relaxation study of β-lactoglobulin A association: Some approaches to the scatchard hydration of globular proteins☆

Abstract A general expression for the concentration-dependent relaxation increment, ( dR dc ) μ , (where R may be R1, the spin-lattice, or R2, the spin-spin relaxation rate of water), has been derived from multicomponent theory for a protein in salt solution. Emphasis is placed on the addition of salt to the aqueous protein to minimize potentially high virial effects due to charge repulsion or to the charge fluctuations predicted by the Kirkwood-Shumaker theory; under conditions where the protein has a high net charge-to-mass ratio the calculation of relaxation increments must employ protein activities in place of concentrations. This treatment was applied to the molecular states of β-lactoglobulin A under associating and nonassociating conditions. In contrast to data in the literature obtained in the absence of salt, where correlation times τc were excessively high and hydration values too low, here values of τc from 2H NMR were in quantitative agreement with those expected from parameters of the known structural states of this protein. With these values, hydrations were obtained by three different ways of calculating the relaxation rate of the bound water from 1H and 2H NMR data. Preferential hydrations, derived from linked functions, for the association of the protein at pH 4.65 were obtained from sedimentation velocity measurements. Combination of the results from the temperature dependence of the deuteron NMR and the linked functions, on the basis of a three-state model, yields slow-tumbling hydration values and correlation times comparable to those obtained from the two-state model. Based on either an isotropic bound-water mechanism or an anisotropic orientational distribution of the water molecules, enthalpies of hydration determined from the three-state model are in accord with those calculated from the two-state model.

[11] Structural interpretation of hydrodynamic measurements of proteins in solution through correlations with X-ray data

Publisher Summary This chapter deals with the structural interpretation of hydrodynamic measurements of proteins in solution through correlations with X-Ray data. Analysis of protein structure by X-ray crystallography has revealed irregular surfaces consisting of many clefts, grooves, and protuberances. Hydrodynamic parameters, which are sensitive to surface characteristics, have been calculated from X-ray crystallographic coordinates and compared with solution values in attempts to answer such questions, but with only marginal success. There appears to be a need for structural information from other nonhydrodynamic sources for use in conjunction with hydrodynamic parameters, so that the combined results may be compared with those from X-ray crystallography. The three hydrodynamic parameters under consideration here are obtained experimentally by the observation of flow under the influence of an applied force. This chapter also presents an alternative procedure for estimating the contribution of hydration to the total frictional coefficient to obtain the contribution of the axial ratio by itself, and thus estimates of geometric parameters from sedimentation coefficients.

[9] Small-angle X-ray scattering

Publisher Summary Among the methods available for the characterization of globular proteins, small-angle X-ray scattering (SAXS) is particularly powerful. This method is capable of yielding the radius of gyration and, when used on the absolute-intensity scale, the molecular weight, hydrated volume, surface-to-volume ratio, and the degree of hydration of a particle in solution. To obtain this information, it is necessary to measure two auxiliary parameters: the concentration and the partial specific-volume of the protein. In addition to the molecular parameters, the thermodynamic parameters of interacting systems can be obtained, such as the association constants of aggregating subunit systems and the degree of preferential interaction of proteins with components of mixed solvent systems. In the case of highly concentrated solutions, in which there are strong long-range intermolecular interactions, SAXS can be used to determine the radial distribution function of the interacting macromolecules in solution. This, in turn, yields the interaction potentials characteristic of the operative forces. At still higher concentrations, distinct bands may appear as the system gradually becomes ordered and X-ray scattering passes over into small-angle X-ray diffraction.

Investigation of differences in the tertiary structures of food proteins by small-angle X-ray scattering

SummaryWith current emphasis in bioengineering on developing new and better structure-function relationships for proteins (e.g., the need for predictability of expected properties prior to cloning), practical and reliable methodology for providing characterization of appropriate features has become of increasing importance. The most potent and detailed technique, X-ray crystallography, has severe limitations: it is so demanding and time-consuming that X-ray coordinates are frequently unavailable for materials of interest; its data relate to static and essentially unhydrated structures, whereas proteins exhibit a variety of dynamic features and function in an aqueous environment; and many proteins of technological importance may never be crystallized. Small-angle X-ray scattering, however, is particularly suitable as a methodology that can provide a substantial number of significant geometric parameters consistent with crystallographic results, that can readily show tertiary structural changes occurring under varying conditions, and that can deal with solutions and gels. Results are presented here from small-angle X-ray scattering investigations of the apo and holo forms of chicken egg-white riboflavin-binding protein, chicken egg-white lysozyme, bovine milk-whey α-lactalbumin and β-lactoglobulin, and bovine ribonuclease. We utilize these observations to compare tertiary structures of these proteins as well as conformational changes in these structures, and to provide a basis for discussion of their physical and biological significance.

Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies

The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 A, the casein micelles. Removal of Ca2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D2O was used instead of H2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, v, and correlation times, tau c, were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and v was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though tau c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.

Protein-Water Interactions from 2H NMR Relaxation Studies: Influence of Hydrophilic, Hydrophobic, and Electrostatic Interactions

The importance of water interactions with proteins in food systems is well documented. A controversy exists, however, as to the nature of these interactions and the effect of protein structural changes on them. To clarify these questions, a method has been developed for determining hydration from the protein concentration-dependence of deuteron resonance relaxation rates. Measurements were made in D2O on beta-lactoglobulin A to study effects of hydrophilic interactions, and on both casein micelles and submicelles to study hydrophobic and electrostatic effects. From the protein concentration-dependent relaxation rates, the second viral coefficients of the proteins were obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, hydrations, upsilon, and correlation times, tau c, were calculated for the protein-associated water; from tau c, the Stokes radius, R, was obtained. Variations in upsilon and R were in accord with known structural changes in molecular states of the proteins. The NMR results are compared with hydrations and structural information derived independently from small-angle X-ray scattering.