Overview

We work on the molecular mechanisms of visual transduction in the vertebrate photoreceptor cells.  Rhodopsin, the dim light receptor, is a prototypic member of the superfamily of the seven-helical surface receptors that couple to the GTP-binding proteins.  Rhodopsin contains covalently bound 11-cis-retinal as the inverse agonist.  Light activation involves isomerization of 11-cis-retinal to the all-trans form, resulting in a conformational change that initiates two biochemical cascades, one leading to sensitization (amplification) and the other to quenching (desensitization).  The system has evolved to sense single photons and at the other extreme, cope with millions of photons in strong light.  The biochemistry in the rod cell ultimately causes closing/opening of the cation conductance channels in the plasma membrane.   This results in hyperpolarization of the cell, activation of the synapses to the subsequent sets of cells in the retina and, eventually, in a signal to the brain.  Experimental approaches in our research are interdisciplinary, including chemical, biochemical, molecular biological and biophysical.

Research Summary

Structure and Function in Rhodopsin:  Like all receptors coupled to G-proteins, rhodopsin contains three distinct domains, the cytoplasmic (intracellular) domain that is involved in all the protein-protein interactions, the transmembrane (TM) domain where the signal transduction begins, by light-catalyzed isomerization of 11-cis-retinal to all-trans retinal, and the intradiscal structural domain.  Our efforts are aimed at understanding structures, specific functions and conformational changes in the three domains.

1. Expression of Rhodopsin Mutant Genes in Mammalian Systems:  We use site-specific mutagenesis in a synthetic rhodopsin gene.  Mutagenesis is by “cassette” replacement, i.e. a short restriction fragment in the synthetic gene is replaced by a synthetic counterpart containing the altered codons.  The gene and its mutants are expressed in monkey kidney (COS 1) cells or in stable cell lines from HEK-293 cells.  The expressed proteins are reconstituted with retinal, purified and characterized.
2. The Intradiscal Domain:  This determines the folding of the opsin to the functional structure. The following pathway for the in vivo folding has been derived. Upon arrival of the nascent protein in the endoplasmic reticulum, the seven helical segments form independently of each other. Antiparallel orientation of the helices forms intradiscal loops. The latter fold to a tertiary globular structure. The folding to this structure is coupled to the packing of the seven helices to form the TM domain.  The folded structure is locked in by the formation of a disulfide bond between Cys110 and Cys187. The critical consequence is the precise formation of the retinal binding pocket. Folding of the intradiscal and the membrane domains allows the cytoplasmic domain to fold into the dark state tertiary structure.
3. Studies of the Conformational Change in the Cytoplasmic Domain of Rhodopsin Upon Light Activation:  We are using a combination of biochemical and biophysical approaches to understand the molecular nature of the conformational change which is at the heart of signal transduction.  Systematic cysteine scanning and pairs of cysteines located at specific sites are being used to deduce the local environment and proximity relationships between different regions of the cytoplasmic domain.  EPR studies demonstrate movement of helices in the TM domain. Mammalian expression systems have now been developed that allow NMR studies by preparation of large amounts of mutants.  Both solution and solid state NMR (MAS) are being used. 19F-NMR offers specific advantages and 19F-labeled cysteines in the cytoplasmic face have shown specific chemical shifts.  Further, 19F-labeled cysteine pairs have shown NOE effects.
4. Point Mutations in Rhodopsin Leading to Retinitis Pigmentosa (Night Blindness):  A large number of naturally occurring point mutations in rhodopsin associated with Retinitis Pigmentosa (RP) are now known.  They occur in all the three domains, cytoplasmic (CP), transmembrane (TM) and intradiscal (ID).  Mutations in both the ID and TM domains cause partial or complete misfolding in rhodopsin.  Misfolding has been shown to be caused by the formation of a disulfide bond different from that in native rhodopsin.  A variety of biochemical studies have shown that the abnormal disulfide bond in misfolded RP mutants is between Cys185 and Cys187 instead of the Cys110-Cys187 bond in native rhodopsin.  Mass spectrometric methods have been applied in direct identification of the above abnormal disulfide bond.
5. Protein-Protein Interactions in Sensitization and Desensitization:  Light-activation initiates protein-protein interactions, the first events in which are the activation of the G-protein, transducin and of rhodopsin kinase which phosphorylates rhodopsin.  Molecular descriptions of the interactions between light-activated rhodopsin and the above proteins are being sought by mapping contacts in the complexes by covalent crosslinking followed by mass spectrometric identification of the contact sites.  Photoactivatable and chemically preactivated crosslinking reagents are being used.

Selected Publications

Chelikani, P., Reeves, P. J., RajBhandary, U. L., and Khorana, H. G. The Synthesis and High Level Expression of a b2-Adrenergic Receptor Gene in a Tetracycline-inducible Stable Mammalian Cell Line. Protein Science, 15:1433-1440 (2006)

Kota, P., Reeves, P. J., RajBhandary, U. L., and Khorana, H. G. Opsin is Present as Dimers in COS1 Cells: Identification of Amino Acids at the Dimeric Interface. Proc. Natl. Acad. Sci. USA 103: 3054-3059 (2006)

Kim, J-M., Hwa, J., Garriga, P., Reeves, P. J., RajBhandary, U. L., and Khorana, H. G. Light-driven Activation of b2-Adrenergic Receptor Signaling by a Chimeric Rhodopsin Containing the b2-Adrenergic Receptor Cytoplasmic Loops. Biochemistry 44: 2284-2292, (2005)

Kim, J-M., Altenbach, C., Kono, M., Oprian, D. D., Hubbell, W. L., and Khorana, H. G. Structural Origins of Constitutive Activation in Rhodopsin: Role of the K296/E113 Salt-bridge. Proc. Natl. Acad. Sci. USA 101: 12508-12513 (2004)

Klein-Seetharaman, J., Yanamala, N. V. K., Javeed, F., Reeves, P. J., Getmanova, E. V., Loewen, M. C., Schwalbe, H., and Khorana, H. G. Differential Dynamics in the G protein-coupled Receptor Rhodopsin Revealed by Solution NMR. Proc. Natl. Acad. Sci. USA 101: 3409-3413 (2004)

Getmanova, E., Patel, A. B., Klein-Seetharaman, J., Loewen, M. C., Reeves, P. J., Friedman, N., Sheves, M., Smith, S. O., and Khorana, H. G. NMR Spectroscopy of Phosphorylated Wild-type Rhodopsin: Mobility of the Phosphorylated C-Terminus of Rhodopsin in the Dark and Light Activation. Biochemistry 43: 1126-1133 (2004)

Reeves, P. J., Callewaert, N., Contreras, R., and Khorana, H. G. Structure and Function in Rhodopsin. High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyl transferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99: 13419-13424 (2002)

Reeves, P. J., Kim, J-M., and Khorana, H. G. Structure and Function in Rhodopsin. A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc. Natl. Acad. Sci. USA 99: 13413-13418 (2002)

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