Rhodopsin, the primary protein constituent of the human retina, has a direct role in human diseases through a variety of point defects in the rhodopsin gene that result in autosomal dominant retinitis pigmentosa (ADRP). ADRP is a form of retinal degeneration, affecting over 100,000 Americans, which can lead to blindness. However, beyond its unfortunate role in human disease, research suggests that rhodopsin offers a powerful tool in the quest for better health. That potential comes from the fact that rhodopsin belongs to a broad class of hepatahelical G protein-coupled receptors (GPCRs) estimated to be the target of about 50% of existing medicines. Early events in the operation of those important receptors, comprising ~5% of the human genome, are a mystery because the non-rhodopsin receptors cannot be synchronously triggered. Rhodopsin offers a way to understand those processes because its natural trigger, light, can be delivered on a sub- nanosecond time scale with modern laser technology. Further, the light absorbing function of rhodopsin allows activation processes to be studied with fast modern optical methods. Understanding the complex early processes in GPCRs should contribute to development of better medications. We propose time-resolved studies of rhodopsin and related visual pigments to characterize the mechanism of GPCR activation with particular emphasis on the integral role of the membrane environment and effects of protein-protein interactions. Time-resolved UV/visible absorbance changes during activation will be measured using an intensified charge coupled device, optical array detector using a flash lamp probe source whose brightness is sufficient for high signal-to-noise ratio sub-microsecond measurements. Data will be analyzed using Matlab routines that allow different mechanistic schemes connecting photointermediates to be compared. The effects of membrane environment on the GPCR activation mechanism will be studied using both liposomes to vary the lipid environment and nanodiscs to preserve membrane-like activation for rhodopsin mutants in a preparation with superior optical properties for measurement. Time-resolved measurements with polarized light, including linear dichroism (LD) and circular dichroism (CD) will be used to obtain more detailed structural information about the activation mechanism. LD will be used to determine transition dipole moment changes during photointermediate progression and to determine whether rhodopsin is a monomer or higher order oligomer in the native disk membrane. Studies of rhodopsin mutants include those in extracellular loop 2, near the chromophore, rhodopsin stabilized by an engineered disulfide linkage and ADRP mutants whose pathology remain unexplained.
Rhodopsin, the primary protein constituent of the human retina, has a direct role in human diseases through a variety of point defects in the rhodopsin gene that result in autosomal dominant retinitis pigmentosa (ADRP). ADRP is a form of retinal degeneration, affecting over 100,000 Americans, which can lead to blindness. Study of rhodopsin activation is directed at understanding ADRP and related diseases outside the eye in rhodopsin's much larger family of G protein coupled receptors.
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