The long term objective of this proposal is to understand the role that lipids play in the function of biological membranes. Specific questions addressed are: What is the role of the highly unsaturated fatty acids (PUFA) found in the phospholipids of ROS membranes? What roles do specific phospholipid headgroups play in the function of ROS? What are the molecular interactions of retinal with amino acid side chains before and after light excitation? 2H-NMR will be used to study protein-lipid interactions by reconstituting rhodopsin with specifically deuterated lipids or by incorporating deuterated lipids into native ROS membranes with phospholipid exchange proteins. Interactions of labeled lipids with rhodopsin and extrinsic membrane proteins will be investigated. 2H-NMR measurements will provide information on the orientational order and motional properties of lipids at various depths in the membranes. The use of deuterated lipids will reduce or eliminate problems of proton spin diffusion and allow application of powerful 1H-NMR methods to be applied. The lateral diffusion of labeled lipids will be measured using a 2H-NMR technique and the average lateral diffusion of all lipids will be measured with 31P NMR. Retinals specifically labeled with 13C will be incorporated into rhodopsin and bacteriorhodopsin in native membranes. Solid state, magic angle spinning NMR methods will be used to map out the detailed interactions between retinal and amino acid side chains of rhodopsin and bacteriorhodopsin. The two point charge model for the bathochromic shift and energy conversion in rhodopsin and bacteriorhodopsin will be investigated. The state of protonation of aspartic and glutamic acids will be studied at key points after light excitation by incorporating 13C labeled Asp and Glu into bacteriorhodopsin. Surface carboxyl groups will be distinguished with paramagnetic broadening reagents. There is physiological evidence that highly unsaturated lipids play an important role in ROS membrane, however, a detailed understanding of this requirement remains obscure. The experiments outlined here are designed to shed new light on the function of PUFA and lipid head groups in native ROS as well as greatly broadening our understanding of the detailed molecular interactions of retinal with the binding pockets in rhodopsin and bacteriorhodopsin.
