Reaction of the purple membrane with a carbodimide

Renthal, Robert; Harris, Gary J.; Parrish, Rob G.

doi:10.1016/0005-2728(79)90009-4

Cited by 37 publications

(21 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previously, Fourier transform infrared spectroscopy (FTIR) studies have indicated the involvement of aspartic acid residues in proton pumping (11)(12)(13)(14) chemical modification experiment has suggested that Asp-115 residue may be involved (15,16). The results reported herein show that, whereas substitution of the four residues Asp-36, -38, -102, and -104 ( Fig.…”

Section: Discussionmentioning

confidence: 49%

Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

Mogi

Stern²,

Marti³

et al. 1988

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

283

269

View full text Add to dashboard Cite

We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, -96, -115, and -212 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their Am=,.X varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85 --Asn mutant showed no detectable proton pumping, the Asp-96 -+ Asn and Asp-212 --Glu mutants showed <10% and the Asp-115 -Glu mutant showed -30% ofthe normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.Bacteriorhodopsin (bR), an integral membrane protein, functions as a light-driven proton pump in Halobacterium halobium. The protein traverses the cytoplasmic membrane seven times and contains one molecule of all-trans-retinal linked as a Schiff base to Lys-216 as the chromophore (Fig. 1). In structure-function studies of this protein, we are investigating the following questions. (i) What is the mechanism of vectorial proton translocation? Does it involve proton conduction through the functional groups of certain specific amino acids in the membrane-embedded regions? (it) What is the nature of the interactions between retinal and the protein and how do these interactions change during different stages of the photochemical cycle? (iii) What is the nature of the interactions between the membrane-embedded segments that lead to a specific folding pathway?By recombinant DNA methods we have carried out a variety of amino acid substitutions that were designed to remove specific functional groups (1-4). All of the mutants bound retinal to regenerate bR-like chromophores, and most showed unchanged light-dependent proton pumping. The mutants could be divided into two groups on the basis of their spectral properties. One group showed essentially the native bR absorption spectrum, whereas the second group showed varying but significant spectral shifts from the native bR spectrum. The only two mutants that showed altered proton pumping were Pro-186 -+ Leu and Tyr-185 --Phe.With the aim of identifying more mutations that affect proton pumping, we have now carried out single substitutions of all the aspartic acid residues (Fig. 1) (it) the mutants Asp-115 -Glu and Asp-212 --Asn showed reduced (35 and 15%, respectively) pumping whereas Asp-212 --Ala was unstable to light and showed no pumping; and (ii,) substitution of Asp-36, -38, -102, -104, and -115 by asparagine residues did not affect proton pumping.We document these findings and discuss their relevance to the mechanism of proton translocation as well ...

show abstract

Section: Discussionmentioning

confidence: 49%

Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

Mogi

Stern²,

Marti³

et al. 1988

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

283

269

View full text Add to dashboard Cite

show abstract

“…We do not see any evidence of a major change in the conformation of bacteriorhodopsin after the carbodiimide reaction (Renthal et al, 1983), or any large alteration in the overall surface charge of purple membrane (Renthal et al, 1979). After periodate oxidation, dansylation and borohydride reduction under conditions similar to those described in Methods, we extracted the purple membrane polar lipids and chromatographed them on silica layers.…”

Section: Uranyl Binding To Purple Membranementioning

confidence: 58%

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence

Renthal¹,

Cha²

1984

Biophysical Journal

View full text Add to dashboard Cite

Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.

show abstract

“…After each experiment the system was calibrated by successive addition of 1 #1 injections of 10 mM HCI and 10 mM NaOH. The BR-coated particle suspension in 25 mM Tris/HCl buffer (pH 7) and 1 mM dithiothreitol was incubated with 5 ~g/ml papain (Serva, Heidelberg, FRG) at 30°C [13]. At different times aliquots were taken and mixed with SDS solution (1% final concentration).…”

Section: Methodsmentioning

confidence: 99%

Oriented incorporation of bacteriorhodopsin into the lipid shell of phospholipid‐coated polymer particles

Rothe¹,

Aurich²,

Engelhardt³

et al. 1990

FEBS Letters

View full text Add to dashboard Cite

Phospholipid monolayers covalently fixed to spherical polymer carriers spontaneously form a stable bilayer-like structure by adsorption of further added phospholipids. Bacteriorhodopsin could be incorporated into the lipid bilayer growing on these particles. From the direction of proton pumping as well as from freeze-fracture electron micrographs and papain digestion, experimental evidence was obtained that the preferred orientation of bacteriorhodopsin within the lipid coat is 'inside-out' (more than 90%). A 'right-side-out' incorporation of bacteriorhodopsin could be demonstrated, if the negative charge density of the carrier surface was lowered by chemical modification.Phospholipid-coated bead; Membrane model; Bacteriorhodopsin

show abstract

Reaction of the purple membrane with a carbodimide

Cited by 37 publications

References 18 publications

Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence

Oriented incorporation of bacteriorhodopsin into the lipid shell of phospholipid‐coated polymer particles

Contact Info

Product

Resources

About