Ordered membrane insertion of an archaeal opsin
            <i>in vivo</i>

Dale, Heather; Angevine, Christine M.; Krebs, Mark P.

doi:10.1073/pnas.140216497

Cited by 48 publications

(32 citation statements)

References 28 publications

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“…This result is consistent with observations relating to folding in vivo, which showed bR helices fold sequentially with helix G folding last (25). bO is inserted sequentially from N-to C-terminal into the membrane via the secretory translocase (46) but only part of the protein is inserted cotranslationally; helices F and G only associate with upstream helices after translation is complete (28). Taken together, the data suggest that a general theme of bR folding in vivo and in vitro is the establishment of a stable core of folded helices toward the N-terminal and the subsequent coalescence of the rest of the structure.…”

Section: Discussionsupporting

confidence: 90%

“…A simple interpretation of these results leads to the hypothesis that the bR transition state will comprise a stable folding core, which minimally includes helix B. Here, we investigate whether this proposed folding core contains elements of another helix of bR, by extending Φ-value analysis to another transmembrane segment of bR, the C-terminal helix G. This helix is of interest because it contains K216, the covalent attachment site for the retinal cofactor, and previous data suggested that unlike helix B, helix G in isolation is not inherently stable (23,24) and that it folds later in the folding pathway in vitro (25)(26)(27) and in vivo (28). Extending our previous successful study we use Ala-scanning mutagenesis to identify suitable mutants and combine kinetic and equilibrium experiments to obtain Φ-values for 12 residues in helix G. Additionally we commence double mutant studies to probe the extent of interhelix hydrogen bonding in the folding transition.…”

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confidence: 99%

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Stable folding core in the folding transition state of an α-helical integral membrane protein

Curnow

Bartolo²,

Moreton³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.

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Section: Discussionsupporting

confidence: 90%

mentioning

confidence: 99%

Stable folding core in the folding transition state of an α-helical integral membrane protein

Curnow

Bartolo²,

Moreton³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…22 In contrast, kinetic radiolabelling approaches revealed the co-translational insertion of the N-terminal portion of the Halobacterium salinarum membrane protein bacterioopsin. 23 Moreover, earlier studies reporting co-sedimentation of SRP (7 S) RNA and bacterioopsin mRNA with membrane-bound polysomes, together with puromycin-induced release of the SRP RNA from the polysomes, 24 as well as more recent studies addressing SRP in H. volcanii 25,26 and other archaeal species, 27 -31 lend further support for the existence of a co-translational mode of protein translocation in Archaea. Direct demonstration of an interaction between ribosomes and the archaeal translocation complex, a central step in a co-translational mode of translocation, remains, however, to be presented.…”

Section: Introductionmentioning

confidence: 83%

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

Ring

Eichler

2004

Journal of Molecular Biology

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“…Under microaerobic conditions, BR is induced ϳ50-fold (6) and forms a twodimensional crystal known as the purple membrane. This system has served as a model for studying key steps in membrane protein biogenesis, including protein insertion into the membrane (7,8) and the assembly of protein-lipid complexes (9, 10). H. salinarum is genetically tractable, and the genome sequence of a closely related organism, Halobacterium sp.…”

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confidence: 99%

brp and blh Are Required for Synthesis of the Retinal Cofactor of Bacteriorhodopsin in Halobacterium salinarum

Peck

Echávarri-Erasun²,

Johnson

et al. 2001

Journal of Biological Chemistry

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Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the ⌬brp strain, bacteriorhodopsin levels are decreased ϳ4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, ␤-carotene, is increased ϳ3.8-fold, whereas retinal is decreased by ϳ3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the ⌬brp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of ␤-carotene increased ϳ5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of ␤-carotene to retinal.Rhodopsins are integral membrane proteins containing seven transmembrane ␣-helices and a covalently bound molecule of retinal. Two distinct rhodopsin families are known: the visual rhodopsins, which bind 11-cis retinal or related compounds and function as photoreceptors in vertebrates (1) and invertebrates (2), and the archaeal rhodopsins, which bind all-trans-retinal and function as light-driven ion pumps and phototaxis receptors in archaea (3). Archaeal rhodopsin orthologs have been found recently in bacteria (4) and fungi (5), suggesting that retinal-based pigments are of widespread significance. Despite their importance, the biogenesis of these molecules is not fully understood. In particular, relatively little is known about how retinal is assembled with the opsin apoprotein in vivo. Thus, a goal in elucidating rhodopsin biogenesis is to identify the cellular factors that mediate the biosynthesis or uptake of retinal, the transport of retinal in the cell, and the binding of retinal to the corresponding opsin.To this end, we have studied the biogenesis of bacteriorhodopsin (BR), 1 a light-driven proton pump in the archaeon Halobacterium salinarum. BR consists of the membrane protein bacterioopsin (BO) and all-trans-retinal. Under microaerobic conditions, BR is induced ϳ50-fold (6) and forms a twodimensional crystal known as the purple membrane. This system has served as a model for studying key steps in membrane protein biogenesis, including protein insertion into the membrane (7,8) and the assembly of protein-lipid complexes (9, 10). H. salinarum is genetically tractable, and the genome sequence of a closely related organism, Halo...

show abstract

Ordered membrane insertion of an archaeal opsin in vivo

Cited by 48 publications

References 28 publications

Stable folding core in the folding transition state of an α-helical integral membrane protein

Stable folding core in the folding transition state of an α-helical integral membrane protein

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

brp and blh Are Required for Synthesis of the Retinal Cofactor of Bacteriorhodopsin in Halobacterium salinarum

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