Dynamic structure of biological and model membranes: analysis by optical anisotropy decay measurement.

Rotational Brownian motion of molecules in membranes can be "visualized" by time-resolved measurement of the decay of anisotropy of various flash-induced optical signals, such as fluorescence, phosphorescence, delayed fluorescence, transient absorption, or fluorescence depletion. The basic principles of the various forms of anisotropy measurement are illustrated in a unified manner. In organized structures such as membranes, rotational motion is restricted in angular range. Methods of analysis of observed optical anisotropy decays for the case of restricted rotation are described; the emphasis is laid on the separate estimation of the two important parameters, the range and rate, that characterize the restricted rotation. Practical aspects of the analytical procedures are also discussed. As an example of application, recent work from the authors' laboratory is reviewed: dynamic structures of lipid hydrocarbon chain region of membranes have been revealed by time-resolved fluorescence depolarization studies. A lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated in model and biological membranes of known compositions. The decay of fluorescence anisotropy indicated that the rod-shaped probe molecules wobbled in the membranes with a wobbling diffusion constant around 0.1 rad2/nsec, presumably reflecting the dynamics of surrounding lipid chains. The effects of temperature, ions, lipid chain unsaturation, cholesterol, and protein on the range and rate of wobbling were examined with model membranes. The dynamic structure of biological membranes was found to be basically similar to that of the bilayer of unsaturated phospholipid; proteins and cholesterol act mainly as barriers that reduce the angular range of wobbling motion.