title: G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins.
abstract: The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.
authors.affiliation: Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
authors.forename: Maria
authors.initials: M
authors.lastname: Rehn
authors.forename: Martina
authors.initials: M
authors.lastname: Moeller
authors.forename: Ulrike
authors.initials: U
authors.lastname: Alexiev
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
authors.forename: George J
authors.initials: GJ
authors.lastname: Turner
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D019295:Computational Biology; D003460:Crystallization; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry; D019617:Halobacterium salinarum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D011522:Protons; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D013053:Spectrophotometry
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D009841:Oligonucleotides; D011522:Protons; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D001436:Bacteriorhodopsins; D004247:DNA; D008239:Lysine