H. Gobind Khorana

Requirement of Rigid-Body Motion of Transmembrane Helices for Light Activation of Rhodopsin

Conformational changes are thought to underlie the activation of heterotrimeric GTP-binding protein (G protein)—coupled receptors. Such changes in rhodopsin were explored by construction of double cysteine mutants, each containing one cysteine at the cytoplasmic end of helix C and one cysteine at various positions in the cytoplasmic end of helix F. Magnetic dipolar interactions between spin labels attached to these residues revealed their proximity, and changes in their interaction upon rhodopsin light activation suggested a rigid body movement of helices relative to one another. Disulfide cross-linking of the helices prevented activation of transducin, which suggests the importance of this movement for activation of rhodopsin.

Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking.

Publisher Summary This chapter presents rhodopsin structure and dynamics at the cytoplasmic face in solution, the comparison between the solution structure and the crystal structure, and the structural changes underlying receptor activation. The data from site-directed spin labeling (SDSL) and disulfide cross-linking together indicate that the crystal and solution structures are very similar at the level of the backbone fold for C1, H8, and the cytoplasmic termination of TM1-TM7. However, substantial differences are seen in C3 and the C-terminal tail, wherein the backbones are flexible on the nanosecond time scale in solution. The chapter also illustrates systematic application of time-resolved SDSL to monitor helix movements, together with the use of chemically modified chromophore structures, is a promising approach to exploring the relationship between chromophore structure, the critical salt bridge, and helix movements leading to activation.

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 H2Ld 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.

[8] Chemical synthesis and cloning of a tyrosine tRNA gene

Publisher Summary This chapter describes the methods used for the construction of a biologically functional suppressor transfer ribonucleic acid (tRNA) gene. For the total synthesis of given deoxyribonucleic acid (DNA)-containing biologically specific sequences, the DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All segments are chemically synthesized, starting with protected nucleosides and mononucleotides. The 5′-hydroxyl groups of the appropriate oligonucleotides are then phosphorylated. The chapter describes the chemical synthesis and cloning of a tyrosine tRNA gene, which involve (1) the synthesis of 126-nucleotide long bihelical DNA corresponding to a known precursor of the tyrosine suppressor tRNA, (2) sequencing of the promoter region and the distal region adjoining the C–C–A end that contains a signal for the processing of the RNA transcript (3) a total synthesis of the 208-base-pair-long DNA, which includes the control elements as well as the Eco RI restriction-endonuclease-specific sequences at the two ends, and (4) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene. The chapter discusses a chemical synthesis of deoxyribooligonucleotides and describes the synthesis of di-, tri-, and tetranucleotides carrying 5′-phosphate groups.

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-β-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.

Location of the Retinal Chromophore in the Activated State of Rhodopsin

Rhodopsin is a highly specialized G protein-coupled receptor (GPCR) that is activated by the rapid photochemical isomerization of its covalently bound 11-cis-retinal chromophore. Using two-dimensional solid-state NMR spectroscopy, we defined the position of the retinal in the active metarhodopsin II intermediate. Distance constraints were obtained between amino acids in the retinal binding site and specific 13C-labeled sites located on the β-ionone ring, polyene chain, and Schiff base end of the retinal. We show that the retinal C20 methyl group rotates toward the second extracellular loop (EL2), which forms a cap on the retinal binding site in the inactive receptor. Despite the trajectory of the methyl group, we observed an increase in the C20-Gly188 (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the β-ionone ring moves to a position between Met207 and Phe208 on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the Cϵ carbons of Lys296 (H7) and Met44 (H1) and between Gly121 (H3) and the retinal C18 methyl group. Helix-helix interactions involving the H3-H5 and H4-H5 interfaces were also found to change in the formation of metarhodopsin II reflecting increased retinal-protein interactions in the region of Glu122 (H3) and His211 (H5). We discuss the location of the retinal in metarhodopsin II and its interaction with sequence motifs, which are highly conserved across the pharmaceutically important class A GPCR family, with respect to the mechanism of receptor activation.

The synthesis and high‐level expression of a β2‐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 β2‐adrenergic receptor (β2‐AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon‐optimized hamster β2‐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 β2‐AR/mg of membrane protein, as measured by saturation binding assay using the β2‐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 β2‐AR stable cell lines using the HEK293S‐TetR‐inducible system, which, after induction, express wild‐type β2‐AR at levels of 220 ± 40 pmol/mg of membrane protein corresponding to 50 ± 8 μg/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 μg/15‐cm plate. This level of expression now makes it feasible to pursue structure–function studies using EPR. Furthermore, scale‐up of β2‐AR expression using suspension cultures in a bioreactor should now allow production of enough β2‐AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.

TIME‐RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR‐185→E: ASP‐96 REPROTONATES DURING O FORMATION; ASP‐85 AND ASP‐212 DEPROTONATE DURING O DECAY

Abstract— The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time‐resolved Fourier transform infrared (FTIR) difference spectroscopy and site‐directed mutagenesis. In an earlier study (Bouschéet al., J. Biol Chem. 266, 11063–11067, 1991) we found that Asp‐96 undergoes a deprotonation during the M→N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp‐85 and Asp‐212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr‐185→Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time‐resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all‐trans O chromophore appear in the time‐resolved FTIR difference spectra in the same time range as the appearance of a red‐shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp‐85, Asp‐96, Asp‐115 and Asp‐212, we conclude that during O formation: (i) Asp‐96 undergoes reprotonation. (ii) Asp‐85 may undergo a small change in environment but remains protonated. (iii) Asp‐212 remains partially protonated. In addition, reisomerization of the chromophore during the N→O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.

Role of group-conserved residues in the helical core of β2-adrenergic receptor

G protein-coupled receptors (GPCRs) belonging to class A contain several highly conserved (>90%) amino acids in their transmembrane helices. Results of mutational studies of these highly conserved residues suggest a common mechanism for locking GPCRs in an inactive conformation and for their subsequent activation upon ligand binding. Recently, a second set of sites in the transmembrane helices has been identified in which amino acids with small side chains, such as Gly, Ala, Ser, Thr, and Cys, are highly conserved (>90%) when considered as a group. These group-conserved residues have not been recognized as having essential structural or functional roles. To determine the role of group-conserved residues in the β2-adrenergic receptor (β2-AR), amino acid replacements guided by molecular modeling were carried out at key positions in transmembrane helices H2–H4. The most significant changes in receptor expression and activity were observed upon replacement of the amino acids Ser-161 and Ser-165 in H4. Substitution at these sites by larger residues lowered the expression and activity of the receptor but did not affect specific binding to the antagonist ligand dihydroalprenolol. A second site mutation, V114A, rescued the low expression of the S165V mutant. Substitution of other group-conserved residues in H2–H4 by larger amino acids lowered receptor activity in the order Ala-128, Ala-76, Ser-120, and Ala-78. Together these data provide comprehensive analysis of group-conserved residues in a class A GPCR and allow insights into the roles of these residues in GPCR structure and function.

High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81

The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8±1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins.