Oxygen Accessibility to Ribonuclease A:  Quantitative Interpretation of Nuclear Spin Relaxation Induced by a Freely Diffusing Paramagnet

Abstract

The nuclear spin relaxation induced by a freely diffusing paramagnetic center provides a direct measure of intermolecular accessibility. A number of factors are involved in a quantitative interpretation of relaxation data including excluded volume effects, solvation differences, and the details of the electron spin relaxation in the paramagnetic center. In the case where the electron relaxation time is short compared with correlation times describing the electron-nuclear coupling, the nuclear spin relaxation rates may be related to the effective local concentration of the paramagnetic center at different locations about the solute of interest. The local concentrations may in turn be related to differences in the local free energies of interaction between the diffusing paramagnet and the cosolute. We demonstrate this approach for the case of ribonuclease A and deduce surface free energy differences for a large number of protein proton sites. We find that the oxygen accessibility is poorly represented by hard-sphere models such as computed solvent or steric accessibility. There is a distribution of local intermolecular interactions with a width of the order of RT that dominates the report of the intermolecular exploration of the protein by this simple solute.

The aqueous reference for ESR oximetry

“…The paramagnetic contribution to the solute proton relaxation rate constant by a freely diffusing paramagnetic relaxation agent through a dipole-dipole coupling has been discussed in detail recently [23,24]. We note that while the electron-nuclear dipolar coupling may dominate the nuclear spin-lattice relaxation rate constant, the electron relaxation induced by oxygen in nitroxides is dominated by spin exchange mechanisms.…”

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

“…Two distinct profiles are obtained depending on whether the immersion depth axis is chosen to be tilted by 258 from the barrel axis as proposed by (40) ther in the form of relaxation rate enhancement or chemical shift perturbations demonstrated for 1 H, 13 C, and 19 F nuclei. In most cases, it has become clear that both surface geometries and local surface free energies play a role in the extent to which the paramagnetic effects are manifested (16). With complementary studies and the use of additional free diffusing paramagnetic probes it may be possible to separately assess surface geometries and free energies (16).…”

“…In most cases, it has become clear that both surface geometries and local surface free energies play a role in the extent to which the paramagnetic effects are manifested (16). With complementary studies and the use of additional free diffusing paramagnetic probes it may be possible to separately assess surface geometries and free energies (16). Finally, the large gradients of oxygen demonstrated in both lipid bilayers and micelles should prove very useful in future NMR studies intended to address immersion depth of membrane embedded amphiphiles or, indeed, topology of membrane proteins.…”

“…This approach may be contrasted with that adopted recently by Teng et al (16) in which the paramagnetic relaxation rate (see later) acting on a given nucleus was expressed in terms of a local oxygen concentration and a steric term, f k , describing the local accessibility of dissolved oxygen. f k could be computed over a lattice of excluded volume by an integral weighted by the inverse sixth power of the distance between the lattice site and the nucleus (16). In contrast, the above steric term, hVi, is proposed to be shorter in range than f k since delocalization or polarization effects are presumably collisionally mediated.…”

“…describes the fluctuations of the I-S dipole coupling, which is parameterized by D and by the electronic spin-lattice and spin-spin relaxation times, T e 1 and T e 2 , as expressed explicitly elsewhere (16). Most importantly, the spectral density terms are not generally expected to vary considerably over the protein surface since the diffusion rate is relatively constant and fast fluctuations of the dipole coupling are dominated by the picosecond timescale spin relaxation which is constant.…”