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H Gobind Khorana

Publications and source records attributed to H Gobind Khorana.

16 recordsLinked to original sources
PubMed2006

Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin.

Membrane proteins play vital roles in every aspect of cellular activities. To study diverse membrane proteins, it is crucial to select the right surfactants to stabilize them for analysis. Despite much effort, little progress has been made in elucidating their structure and function, largely because of a lack of suitable surfactants. Here we report the stabilization of a G protein-coupled receptor bovine rhodopsin in solution, using a new class of designer short and simple peptide surfactants. These surfactants consist of seven amino acids with a hydrophilic head, aspartic acid or lysine, and a hydrophobic tail with six consecutive alanines. These peptide surfactants not only enhance the stability of bovine rhodopsin in the presence of lipids and the common surfactants n-dodecyl-beta-D-maltoside and octyl-D-glucoside, but they also significantly stabilize rhodopsin under thermal denaturation conditions, even after lipids are removed. These peptide surfactants are simple, versatile, effective, and affordable. They represent a designer molecular nanomaterial for use in studies of diverse elusive membrane proteins.

Amino Acid Sequence↗
PubMed2006

Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface.

Rhodopsin in the disk membranes of rod outer segments serves as the dim-light photoreceptor and is a prototypic member of a G protein-coupled receptor family. Electron and atomic-force microscopy indicate that rhodopsin is present as dimers in the native membranes. Here, we have expressed the protein, opsin, in COS1 cells and have studied its molecular state by using FRET and by intermolecular cross-linking after site-directed cysteine mutagenesis. To observe FRET, the ends of the genes corresponding to the N termini of the cyan or yellow fluorescent proteins were fused to the ends of the genes corresponding to the C terminus of the opsin and the resulting fused genes were expressed in COS1 cells. The emission spectra in situ of the expressed proteins were recorded, and FRET was then calculated. The result indicated intermolecular interaction between opsin molecules in COS1 cells. To identify the amino acids involved in the interaction, those predicted by molecular modeling to be at the dimer interface were mutated one at a time to cysteine, and dimer formation was measured by the rate of disulfide bond formation in the presence of cupric orthophenanthroline. The mutants W175C and Y206C formed the dimers most rapidly, showing that the two amino acids were at the dimer interface.

Amino Acid Sequence↗
PubMed2006

The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line.

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.

Adrenergic beta-2 Receptor Antagonists↗
PubMed2005

Changes in interhelical hydrogen bonding upon rhodopsin activation.

Hydrogen bonding interactions between transmembrane helices stabilize the visual pigment rhodopsin in an inactive conformation in the dark. The crystal structure of rhodopsin has previously revealed that Glu122 and Trp126 on transmembrane helix H3 form a complex hydrogen bonding network with Tyr206 and His211 on H5, while the indole nitrogen of Trp265 on H6 forms a water-mediated hydrogen bond with Asn302 on H7. Here, we use solid-state magic angle spinning NMR spectroscopy to probe the changes in hydrogen bonding upon rhodopsin activation. The NMR chemical shifts of 15N-labeled tryptophan are consistent with the indole nitrogens of Trp126 and Trp265 becoming more weakly hydrogen bonded between rhodopsin and metarhodopsin II. The NMR chemical shifts of 15N-labeled histidine show that His211 is neutral; the unprotonated imidazole nitrogen is not coordinated to zinc in rhodopsin and becomes more strongly hydrogen bonded in metarhodopsin II. Moreover, measurements of rhodopsin containing 13C-labeled histidine show that a strong hydrogen bond between the side-chain of Glu122 and the backbone carbonyl of His211 is disrupted in metarhodopsin II. The implications of these observations for the activation mechanism of rhodopsin are discussed.

Cell Line↗
PubMed2005

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops.

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.

Amino Acid Sequence↗
PubMed2004

Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge.

The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q/M257Y and G90D/M257Y produce strong activation of the receptor in the dark state. In the E113Q/M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation.

Animals↗
PubMed2004

Identification of core amino acids stabilizing rhodopsin.

Rhodopsin is the only G protein-coupled receptor (GPCR) whose 3D structure is known; therefore, it serves as a prototype for studies of the GPCR family of proteins. Rhodopsin dysfunction has been linked to misfolding, caused by chemical modifications that affect the naturally occurring disulfide bond between C110 and C187. Here, we identify the structural elements that stabilize rhodopsin by computational analysis of the rhodopsin structure and comparison with data from previous in vitro mutational studies. We simulate the thermal unfolding of rhodopsin by breaking the native-state hydrogen bonds sequentially in the order of their relative strength, using the recently developed Floppy Inclusion and Rigid Substructure Topography (FIRST) method [Jacobs, D. J., Rader, A. J., Kuhn, L. A. & Thorpe, M. F. (2001) Proteins 44, 150-165]. Residues most stable under thermal denaturation are part of a core, which is assumed to be important for the formation and stability of folded rhodopsin. This core includes the C110-C187 disulfide bond at the center of residues forming the interface between the transmembrane and the extracellular domains near the retinal binding pocket. Fast mode analysis of rhodopsin using the Gaussian network model also identifies the disulfide bond and the retinal ligand binding pocket to be the most rigid region in rhodopsin. Experiments confirm that 90% of the amino acids predicted by the FIRST method to be part of the core cause misfolding upon mutation. The observed high degree of conservation (78.9%) of this disulfide bond across all GPCR classes suggests that it is critical for the stability and function of GPCRs.

Amino Acid Sequence↗
PubMed2004

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR.

G protein-coupled receptors are cell-surface seven-helical membrane proteins that undergo conformational changes on activation. The mammalian photoreceptor, rhodopsin, is the best-studied member of this superfamily. Here, we provide the first evidence that activation in rhodopsin may involve differential dynamic properties of side-chain versus backbone atoms. High-resolution NMR studies of alpha-(15)N-labeled receptor revealed large backbone motions in the inactive dark state. In contrast, indole side-chain (15)N groups of tryptophans showed well resolved, equally intense NMR signals, suggesting restriction to a single specific conformation.

Amino Acid Sequence↗
PubMed2004

NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light activation.

Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.

Amino Acid Sequence↗
PubMed2004

Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin.

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.

Animals↗
PubMed2002

Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes.

We report on preparation of rhodopsin proteoliposomes with the cytoplasmic domain of rhodopsin facing the exterior of the proteoliposomes. Rhodopsin purified from rod outer segments of bovine retinae by immunoaffinity chromatography in octyl glucoside was reconstituted into liposomes prepared from soybean phospholipids by detergent dialysis. The orientation of rhodopsin in the liposomes was determined by susceptibility of its C terminus to papain and the endoproteinase, Asp-N, followed by SDS/PAGE, which showed that the cytoplasmic domain in at least 90% of rhodopsin faced the exterior of the proteoliposomes. By using escape of (32)P-KP(i) encapsulated in the proteoliposomes as the assay, the half-life of the proteasomes was approximately 8 days. After light activation, rhodopsin in proteoliposomes showed the rate of decay of metarhodopsin II and the initial rate of transducin activation comparable with the rates of rhodopsin in rod outer segment membranes. This finding demonstrates the functional capability of rhodopsin in proteoliposomes for kinetic studies of protein-protein interactions.

Animals↗
PubMed2002

Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants.

Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H(2)L(d) promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.

Animals↗
PubMed2002

Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line.

An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI(-) HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI(-) stable cell line showed it to be Man(5)GlcNAc(2). In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI(-)-inducible cell line at levels comparable with those obtained with WT rhodopsin.

Animals↗
PubMed1991

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin.

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

Bacteriorhodopsins↗