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.