Biophysical studies of integral membrane proteins: 1. A segmented approach to determine the three-dimensional structures of integral membrane proteins 2. Thermal denaturation studies of bovine rhodopsin
Date of Completion
Chemistry, Biochemistry|Biophysics, General
Integral membrane proteins play a major role in several physiological processes. Owing to the presence of parts of these proteins within the biological membranes, they pose a great difficulty to work with. Amidst the problems involved in working with these proteins, several biophysical and biochemical studies have been carried out on these proteins to understand the principles underlying their structure and function. The work described in this thesis is an attempt made in the pursuit of understanding these principles. ^ The first part of the thesis focuses on the structural studies of the integral membrane proteins, bovine rhodopsin, bacteriorhodopsin, and the human β2 adrenergic receptor. A segmented approach to determine the three-dimensional structure of the seven transmembrane helical receptors is developed and successfully applied to determine the three-dimensional structure of bacteriorhodopsin. Later this approach has been used to determine the structures of bovine rhodopsin in both the dark-adapted and the light-adapted states. The structures obtained using this approach are comparable to the medium resolution crystal structures. In addition, structure studies of the helix 8 of the human β2 adrenergic receptor are described in chapter 4. Helix 8 of the β2 adrenergic receptor exhibits conformational flexibility, which may have implications in the receptor activation. This kind of behavior of helix 8 has been observed in several other GPCRs including the bovine rhodopsin. ^ The second part of the thesis focuses on the thermal denaturation studies of bovine rhodopsin, a photoreceptor present in the rod outer segment disk membranes. Rhodopsin aggregates on heating to higher temperatures, exhibiting a thermal transition at 70°C. The thermal denaturation of rhodopsin in the disk membranes is shown to be under kinetic control using both DSC and CD. Later, it is shown that the cytoplasmic loops 2 and 3 play a significant role in the stabilization of the receptor. In addition, the studies described in chapter 8 indicate that the disk membrane cholesterol modulates the thermal stability of rhodopsin in the disk membranes. ^
Katragadda, Madan, "Biophysical studies of integral membrane proteins: 1. A segmented approach to determine the three-dimensional structures of integral membrane proteins 2. Thermal denaturation studies of bovine rhodopsin" (2003). Doctoral Dissertations. AAI3078043.