Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing

Fimmel, Sabine; Choli, Theodora; Dencher, Norbert A.; Büldt, Georg; Wittmann‐Liebold, Brigitte

doi:10.1016/0005-2736(89)90120-x

Cited by 35 publications

(23 citation statements)

References 37 publications

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“…Both of these conjugation sites are at the extracellular side of the PM ( Fig. 1) and freely exposed at the BR surface (4,21). It has been reported that biotin N-hydroxysuccinimide ester labels (under conditions very similar to those applied here) solely an extracellular lysine residue (15).…”

Section: Resultsmentioning

confidence: 63%

“…The carboxamide formed by FSE is more stable than the thiourea formed by FITC (20 site-specific proteolysis with chymotrypsin, papain, or proteinase K were highly fluorescent on the gel. From the previously determined amino acid sequence of these fluorescent peptide fragments (21,22), the fluorescein conjugation site(s) in BR could be unambiguously identified. For example, the chymotryptic C2 fragment (amino acids 1-71, ref.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

Heberle

Dencher

1992

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

162

164

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Light-induced HI release and reuptake as well as surface potential changes inherent in the bacteriorhodopsin reaction cycle were measured between 10'C and 5OC. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle.During the L5,50 -M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescei or by the potentiometric probe 4-{[2-(di-n-butylamino)-6-naphthyllvinyl}-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrocbemical H+ gradients.A challenge in membrane research is the understanding of generation, maintenance, and utilization of electrochemical H+ gradients. Especially the translocation of H+ across integral membrane proteins and subsequent H+ transfer into the aqueous bulk phase are less than well understood. Analysis of vectorial H+ transfer reactions is hampered by strong interactions of H+ with the membrane surface-i.e., with lipid head groups and exposed amino acids (1). These interactions will severely influence the rate of diffusioncontrolled transfer of H+ into the aqueous bulk phase (2). Therefore, it is necessary to monitor the kinetics of H+ transfer directly at the membrane surface, e.g., close to the H+ ejection site of an integral membrane protein (3).Bacteriorhodopsin (BR) in the purple membrane (PM) of Halobacterium halobium is a very attractive system for the investigation of molecular steps in light-driven vectorial H+ translocation. Although BR is an integral membrane protein (26,500 Da) with seven transmembrane a-helices, its structure is known at 3.5-to 7.8-A resolution, including the approximate position of many of the 248 amino acids (4). From the position of the chromophore retinal (5, 6), bound to Lys-216, the location of the protonated nitrogen of the Schiff base that is part of the active center of this transport protein can be inferred. To correlate H+ ejection kinetics with the corresponding spectroscopic intermediate(s) of the BR photocycle (7,8), with protonation changes of amino acids (9-12), and with alterations of the tertiary structure of the protein in the vicinity of the chromophore (13, 14), H+ transfer was monitored with the optical pH indicator fluorescein covalently linked to the extracellular surface of BR, at Lys-129. Concomitant surface potential changes were reflected by absor...

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Section: Resultsmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

Heberle

Dencher

1992

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

162

164

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“…Significantly, in retrospect (since the structure of this receptor is now known 28 ) transmembrane peptide bonds were protected from cleavage since no digests occurred to the protein located in the plane of the bilayer and digestion did not apparently compromise native proteinlipid bilayer interactions. Similar to the studies with bacteriorhodopsin, 29 we assume that limited proteolysis of GlyR would define the limits of TM segments and refine the topology of the receptor. Recent advances in mass spectrometry (MS) allow one to conduct these types of studies on proteins of lower abundance (relative to bacteriorhodopsin) since MS is exquisitely sensitive (typically in the pico-to femtomolar range).…”

Section: Limited Proteolysismentioning

confidence: 96%

Glycine receptors: Lessons on topology and structural effects of the lipid bilayer

Cascio

2002

Biopolymers

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The members of the superfamily of nicotinicoid receptors, sometimes referred to as the ligand-gated ion channel superfamily (LGICS), are essential mediators in the propagation of electrical signals between cells at neuronal and neuromuscular synapses. Given the significant sequence and proposed topological similarities between family members, the structural architecture of any one of these neuroreceptors is believed to be archetypic for the family of ligand-gated channels. We have focused our biophysical studies on the glycine receptor (GlyR) since homomeric expression of just the alpha1 chain of the receptor is sufficient to reconstitute native-like activity when expressed in heterologous cells, and we have successfully overexpressed and purified relatively large quantities of this receptor. Our CD data suggests that the historical four transmembrane helix topology model for nicotinicoid receptors may be erroneous. Proteolytic studies as well as chemical modification studies coupled with mass spectroscopy (MS) have provided additional evidence that this model may be inappropriate. While we suggest a novel topological model for the superfamily of nicotinicoid receptors, the absence of high resolution data for the transmembrane regions of these ion channels precludes further refinement of this model. In addition, we observe structural changes in the recombinant alpha1 GlyR as a function of bilayer composition, suggesting that these receptors may be dynamically modulated by cellular control over the properties of the plasma membrane.

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“…Against hemo globin it is about 6 times more active than pronase and about 3 times more active than bovine trypsin [6], Furthermore, proteinase K has been used extensively to digest proteins or inactivate ribonucleases [7][8][9][10], To minimize the risk that this enzyme might damage RPE cells, we used the lowest concentration (0.1 %) of proteinase K that was effective in isolating RPE cells from intact rat eyeballs.…”

Section: Discussionmentioning

confidence: 99%

“…One of the serine proteases, proteinase K, is a so-called strong protease [6] that has been used to extract mRNA from cells [7], for rapid purification of ferritin from red blood cells [8] or of lipopolysaccharide from bacteria [9] and to establish the sequence of amino acids [10]. Proteinase K has also been used in ophthal mologic research, to isolate the retina and RPE cells from neonatal mice [11],…”

Section: Introductionmentioning

confidence: 99%

A Rapid Method for Isolation of Retinal Pigment Epithelial Cells from Rat Eyeballs

et al. 1995

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A new, simple method for isolating retinal pigment epithelial (RPE) cells from rat eyeballs was developed: incubation of the eyeballs in 0.1% proteinase K solution. This method yielded an average of 4 × 10⁴ viable RPE cells from each Sprague-Dawley rat eyeball in 1 h. These RPE cells were hexagonal and had cytokeratin in their cytoplasm and numerous microvilli on their surface, similar to RPE cells evaluated in vivo. The results of this study thus show that our method for incubating eyeballs in proteinase K can provide good-quality RPE cells in sufficient quantities for study in a short time.

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Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing

Cited by 35 publications

References 37 publications

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

Glycine receptors: Lessons on topology and structural effects of the lipid bilayer

A Rapid Method for Isolation of Retinal Pigment Epithelial Cells from Rat Eyeballs

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