Rotational diffusion of mitochondrial ADP/ATP carrier studied by saturation-transfer electron spin resonance
Laszlo I. Horvath, Anton Munding, Klaus Beyer, Martin Klingenberg, Derek Marsh
The rotational mobility of the mitochondrial A D P / A T P carrier has been studied solubilized in Triton micelles, reincorporated in phospholipid liposomes, and in mitochondria. Spin-labeled analogues of the noncovalent inhibitors carboxyatractyloside and atractyloside were found to be strongly immobilized when bound to the carrier [Munding, A., Beyer, K., & Klingenberg, M. (1983 ) Biochemistry 22, 1941 such that saturation-transfer electron spin resonance spectroscopy could be used to study
... e rotational motion of the protein. Spin-labeled maleimide covalently bound to the carrier was found to have independent segmental motion and hence to be unsuitable for studies of protein rotation. The ESR spectra of the carboxyatractyloside and atractyloside spin labels were found to contain a second component from label in the lipid or detergent, necessitating the use of the saturation-transfer ESR integral method [HorvBth, L. I., & Marsh, D. (1983) J . Magn. Reson. 54, 363-3731 to analyze the rotational motion of the label component bound to the protein. Effective rotational correlation times obtained from integration of the high-field region of the spectrum were lower than those obtained from the total spectral integral, indicating strongly anisotropic rotational diffusion of the carrier in the vesicular and membrane systems, with the spin-label z axis oriented preferentially perpendicular to the rotation axis. Using the results of spectral line-shape simulations for highly anisotropic rotation [Robinson, B. H., & Dalton, L. R. (1980) J . Chem. Phys. 72, 1312-13241, it was estimated that the correlation times for the axial rotational diffusion of the carrier were 7~11 = 1-5 p s in phospholipid liposomes and 2-20 p s in mitochondria, at temperatures of 30 and 0 OC, respectively. These values are upper estimates. Comparison with theoretical estimates of the rotational correlation time based on hydrodynamic models suggests that the carrier protein is a dimer when reincorporated in phospholipid liposomes and in detergent and that it undergoes slower rotational diffusion due to interaction with other proteins in the mitochondrial membrane.