ANALYSIS OF SATURATION-TRANSFER ELECTRON-PARAMAGNETIC-RESONANCE SPECTRA OF A SPIN-LABELED INTEGRAL MEMBRANE-PROTEIN, BAND-3, IN TERMS OF THE UNIAXIAL ROTATIONAL DIFFUSION-MODEL

Algorithms have been developed for the calculation of saturation trans fer electron paramagnetic resonance (ST-ERR) spectra of a nitroxide sp in-label assuming uniaxial rotational diffusion, a model that is frequ ently used to describe the global rotational dynamics of large integra l membrane proteins. One algorithm explicitly includes terms describin g Zeeman overmodulation effects, whereas the second more rapid algorit hm treats these effects approximately using modified electron spin-lat tice and spin-spin relaxation times. Simulations are presented to demo nstrate the sensitivity of X-band ST-ERR spectra to the rate of uniaxi al rotational diffusion and the orientation of the nitroxide probe wit h respect to the diffusion axis. Results obtained by using the algorit hms presented, which are based on the transition-rate formalism, are i n close agreement with those obtained by using an eigenfunction expans ion approach. The effects of various approximations used in the simula tion algorithms are considered in detail. Optimizing the transition-ra te formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treat ment of nonaxial A- and g-tensors. The algorithms presented here are u sed to perform nonlinear least-squares analyses of ST-EPR spectra of t he anion exchange protein of the human erythrocyte membrane, band 3, w hich has been affinity spin-labeled with a recently developed dihydros tilbene disulfonate derivative, [N-15,H-2(13)]-SL-H(2)DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocyt es undergo rotational diffusion about the membrane normal axis at a ra te consistent with a band 3 dimer.