Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates

Abstract

The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18 degrees C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation times (tau 0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs omega e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of tau 0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-alpha-glycero-phosphocholine (GPC)] to a high of 12 ns (alpha-ATP) at 10 degrees C, whereas at 18 degrees C the range was from ca. 1 to 9 ns. For the nucleus the tau 0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10 degrees C; at 18 degrees C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-alpha-glycerol phosphate) at 18 degrees C evidence was obtained for binding and subsequent exchange with solid like protein domains. The diversity in tau 0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of tau 0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18 degrees C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs omega e curves to yield the

Interactions of Chaperone α-Crystallin with the Molten Globule State of Xylose Reductase

“…This acts to reduce the hydrophobic interactions and thus the affinity of the chaperone for the substrate protein further allowing reconstitution of the active XR. Evidence for the ATP-␣-crystallin binding has earlier been provided by 31 P NMR spectroscopy (37) and fluorescence studies (38). ATP failed to influence the tryptophanyl fluorescence of XR in 1.4 M GdmCl (XR-m) indicating inability of the adenine nucleotide to bind XR-m (data not shown).…”

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment

“…The minimum average correlation time for lens protein reorientational motion is 100 ns at 18°C in both cortical and nuclear homogenates (53). The values obtained for the ions depend, as noted above, on the type of model used to analyze the data.…”