Comparisons of the relative effects of polyhydroxyl compounds on local versus long-range motions in the mitochondrial inner membrane. Fluorescence recovery after photobleaching, fluorescence lifetime, and fluorescence anisotropy studies

Abstract

This laboratory has been interested in understanding the relationship between molecular motion and electron transport rates in the mitochondrial inner membrane. We have previously noted a sucrose-induced decrease in both multicomponent electron transport rates and lateral diffusion of redox components. The decreases in lateral diffusion and the related mobile fraction of redox components were greater than expected from hydrodynamic theory. In this report we sought to understand how the presence of increasing aqueous concentrations of polyhydroxyl agents affect short-range motions in different regions of the inner membrane bilayer, frequently expressed in terms of ‘viscosity’ and order, compared to lateral diffusion. Fluorescence recovery after photobleaching was used to monitor long-range phospholipid and integral protein diffusion. Multifrequency fluorescence lifetime and steady-state fluorescence anisotropy techniques were used to monitor local dynamics of diphenylhexatriene (DPH) and trimethylaminodiphenylhexatriene (TMA-DPH). Light scattering corrections were found to be essential for inner membrane measurements by the latter two techniques. DPH and TMA-DPH each exhibited two-lifetime components. Generally, increasing the aqueous concentration of polyhydroxyl agents decreased the average DPH lifetime and increased the average TMA-DPH lifetime. In general, under the same conditions fluorescence anisotropies increased. Our results indicated that changes in the rotational diffusion coefficient, microviscosity and order were being induced at both the phospholipid headgroup and in the acyl chain regions of the membrane bilayer. Our results suggest that these changes may be due in part to induced changes in the interaction and distribution of water with membranes. Long-range lateral diffusion was found to be significantly retarded by increasing concentrations of polyhydroxyl agents. We conclude that the discrepancies between bulk viscosity predicted decreases in long-range diffusion may result, in part, from the aforementioned membrane/water interactions. We also note an apparent qualitative relationship between long-range lateral diffusion reported diffusion coefficient with local TMA-DPH reported rotational diffusion coefficient and apparent microviscosities.