Mapping Oxygen Accessibility to Ribonuclease A Using High-Resolution NMR Relaxation Spectroscopy

Abstract

Paramagnetic contributions to nuclear magnetic spin-lattice relaxation rate constant induced by freely diffusing molecular oxygen measured at hundreds of different protein proton sites provide a direct means for characterizing the exploration of the protein by oxygen. This report focuses on regions of ribonuclease A where the rate constant enhancements are either quite large or quite small. We find that there are several regions of enhanced oxygen affinity for the protein both on the surface and in interior pockets where sufficient free volume permits. Oxygen has weak associative interactions with a number of surface crevices that are generally between secondary structural elements of the protein fold. Several regions near the surface have higher than expected accessibility to oxygen indicating that structural fluctuations in the protein provide intermolecular access. Oxygen penetrates part of the hydrophobic interior, but affinity does not correlate simply with hydrophobicity indices. Oxygen is excluded from regions of high interior packing density and a few surface sites where x-ray diffraction data have indicated the presence of specific hydration with high occupancy.

Molecular oxygen as a paramagnetic NMR probe of protein solvent exposure and topology

“…Moreover, one need not be concerned with deuteration levels and the effects of spin-diffusion in the case of these paramagnetic shifts. Modest oxygen-induced 1 H chemical shift perturbations have been previously reported in protein studies (12), and 19 F chemical shift perturbations as high as 4.0 ppm at 100 bar oxygen partial pressures have been observed and subsequently used to study membrane immersion depth (13) and protein solvent exposure (14). Measurable 13 C paramagnetic shifts (roughly 0.4 ppm or less) at oxygen partial pressures of 30 and 60 bar were also reported and interpreted in terms of protein structure and solvent exposure.…”

“…Further evidence for the burial of the Trp36 indole was presented in a recent 19 F and 1 H NMR spectro- scopic study of z3-fluorotryptophan substituted drkN SH3 domain 10 ( Fig. 5).…”

“…The effect of O 2 on chemical shifts of 1 H, 19 F, and 13 C nuclei provides a perspective of protein topology and solvent exposure, and the resulting paramagnetic shifts are straightforward to analyze. Chemical shift perturbations may be directly related to local oxygen concentrations, while they are not explicitly dependent on O 2 exchange dynamics in the weak collision limit (i.e.…”

“…Thus, R 1p is acutely sensitive to local topology (i.e., f and b above). Note that since the paramagnetic rates depend on the square of the gyromagnetic ratio, relatively large (relaxation) effects may be seen on 1 H and 19 F nuclei (8,11,12,(18)(19)(20) …”

The Reaction of Hydrogen Atoms with Methionine Residues: A Model of Reductive Radical Stress Causing Tandem Protein–Lipid Damage

“…In fact, previous evaluations of the accessibility of RNase A amino acids to oxidizing agents and oxygen indicated Met29 or Met30 as the most-exposed methionines of the protein. [34,35] …”