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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY006913-02
Application #
3263625
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1986-06-10
Project End
1988-05-31
Budget Start
1987-06-01
Budget End
1988-05-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Montana State University Bozeman
Department
Type
Schools of Arts and Sciences
DUNS #
City
Bozeman
State
MT
Country
United States
Zip Code
59717