Title

Structural and biophysical studies on the retinal rod outer segment membrane proteins peripherin/rds and rhodopsin

Date of Completion

January 2008

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Retinal rod photoreceptor cells are specialized neurons in the vertebrate retina that initiate the visual response. Disease-linked mutations in the two rod outer segment (ROS) membrane proteins, rhodopsin and peripherin/rds, lead to various forms of retinal degeneration. Therefore, understanding the function of these proteins and how that function is supported by structure is key to unraveling the mechanism of both visual responses and retinal degenerative diseases. ^ The cytoplasmic C-terminal domain of the ROS disk membrane, tetraspannin protein peripherin/rds (PerCter), binds to membranes, adopts a helical conformation, and promotes membrane fusion during ROS renewal. Using nuclear magnetic resonance (NMR) and fluorescence spectroscopy, the structure of the PerCter in solution and when bound to the membrane mimetic dodecylphosphocholine micelles is characterized. These studies reveal that PerCter is unstructured in solution and becomes helical when bound to the micelles. The fusion-active sequence within PerCter has a motif similar to the influenza hemagglutinin viral fusion peptide. Together these results suggest that peripherin/rds may function analogously to viral fusion proteins. ^ A novel calcium-dependent interaction between PerCter and calmodulin (CaM) is identified using homology modeling, pull-down assays, and fluorescence spectroscopy. It is shown that Ca2+/CaM binding to PerCter inhibits its ability to promote membrane fusion, which suggests calmodulin may participate in regulating ROS renewal. Ca2+/CaM binding to PerCter is the first identified interaction between CaM and a tetraspannin. A foundation for determining the atomic structure of Ca2+/CaM bound to the PerCter using NMR spectroscopy is established. These results have a significant impact on understanding the role of the peripherin/rds in vivo and its fusogenic function. ^ Recent reports suggested that the G-protein coupled receptor rhodopsin might form dimers or high-order oligomers in ROS disk membranes. These reports contradict the long-standing monomeric model for rhodopsin in disk membranes. The results reported here from an extensive differential scanning calorimetry study utilizing native ROS disk membranes, and cross-linked disk membranes that are known to contain rhodopsin dimers, support a monomeric model for rhodopsin. Furthermore, these studies provide insight into molecular consequences of thermal-induced membrane protein denaturation, which may ultimately lead to a greater ability to manipulate membrane protein stability. ^