Photoreactive labeling of M13 coat protein in model membranes by use of a glycolipid probe.

Abstract

Coliphage M13 coat protein in synthetic bilayer membranes was labeled by use of 12,(4-azido-2-nitrophenoxy)stearoyl[I-14Clglucosamine, a photoreactive glycolipid probe that spontaneously inserts into membranes. In this model system, the probe preferentially labeled the proteins over the lipids. Experiments designed to test the probe's restriction to integral membrane proteins revealed that extrinsic proteins as well as external peptide fragments of integral membrane proteins were not accessible to the photogenerated nitrene on the fatty acid chain. Only integral membrane peptides were labeled by the membrane-bound probe. These results indicate that protein labeling can be effected specifically from within the hydrocarbon milieu of a model membrane system. Recently, we described the synthesis of several photoreactive probes designed for the high-resolution mapping of membrane proteins (1, 2). In addition, we demonstrated the ability of one of these probes, 12-(4-azido-2-nitrophenoxy)stearoyl[1-'4C]-glucosamine, designated 12-APS-GlcN, to specifically label the envelope proteins of Newcastle disease virus (3) and to follow the transmembrane dynamics involved in cholera toxin activity (4). These studies prompted further investigation into the nature of the interaction of the probe with membrane proteins in an effort to define the region of the probe's reactivity and to verify the probe's restriction to integral membrane proteins. Toward this goal, we have constructed phospholipid vesicles containing the 5000-dalton coat protein of the coliphage M13, which orients itself unidirectionally across the membrane bilayer under appropriate conditions (5). Because the amino acid sequence as well as the hydrophobic region of this protein are known, it should be possible to determine the position of the probe in the membrane by using the hydrophobic amino acids of the protein as a gauge of membrane depth.In this report, we show that M13 coat protein incorporated into dimyristoyllecithin (DML) vesicles is indeed labeled by the photoreactive probe (12-APS-GlcN) upon irradiation. Furthermore, evidence is presented that indicates that the probe does not label externally added coat protein, nor does it label amino acid residues of the protein that are shown to be outside of the phospholipid bilayer by trypsin cleavage experiments.MATERIALS AND METHODS DML was obtained from Sigma or Calbiochem; trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was purchased from Worthington. Both were used without further purification.Preparation of M13 Coat Protein. M13 virus was grown and isolated as described by Wickner (6) with slight modifications. Escherichia coli strains K-37 and LA-9 were used for growing unlabeled and [3H]proline-labeled virus, respectively. The bacteria were cultured either in YT medium or in M63 minimal medium (7) to optimize the uptake of label. The M13 coat protein was extracted from whole virus by established procedures (8) and stored at either +40C or -20°C in 50 mM Tris buffer (pH...

Identification of membrane proteins of human blood platelets with a hydrophobic photolabel

“…In contrast to Hu and Wisnieski [13], we have found that coupling of 12-APS-GA to dimyristoyl-PC in vesicles is very limited (unpublished observation). Our observations agree with the notion that aryl nitrenes do not easily insert into C-H bonds [20,23].…”

“…1) ensures an anchoring of the nitrene-generating group in the membrane core by its attachment to the fatty acyl chain, while the carbohydrate moiety is meant to restrict the probe to the outer leaflet of sealed membrane systems [8][9][10]. In the case of coliphage M13 coat protein reconstituted in PC vesicles, evidence was provided that this probe coupled to the integral membrane peptides and not to the amino acid residues exposed on the outside of the bilayer [13,17]. Here we have used 12-APS-GA to identify the integral membrane proteins of human blood platelets.…”

“…In order to determine the amount of probe coupled to membrane proteins after irradiation, an aliquot of the packed cells (5% of total) was analyzed by TLC using chloroform/methanol/ ammonia (65 : 35 : 5, v/v) as described by Hu and Wisnieski [13]. The plates were scanned for radioactivity using a thin-layer scanner (Panax RTLS-IA).…”

Enhanced complement-mediated lysis of type III paroxysmal nocturnal hemoglobinuria erythrocytes involves increased C9 binding and polymerization.

“…At different times, 0.5 ml of reaction mixture was removed, and the reaction was stopped by addition of 0.5 ml of ice-cold P1/NaCl. The (23). Complement-lysed cells were incubated with the photo-reagent and irradiated to effect crosslinking between inserted proteins and the radioactive photoprobe.…”

Labelling of the integral proteins of sarcoplasmic-reticulum membranes

“…In fact they have been shown to label only the hydrophobic tail of cytochrome b5 (Bisson et al, 1979), and none of the subunits of the hydrophilic F1 domain of the mitochondrial and the Escherichia coli ATP synthetase were cross-linked by arylazidophospholipids (Montecucco et al, 1983). Moreover, only intrinsic peptide fragments of the coliphage Ml 3 coat protein were derivatized by an azidoglycolipid probe (Hu & Wisnieski, 1979). In the case of the cytochrome oxidase subunit II and subunit b of the ATP synthetase, the precise sites of cross-linking with azidophospholipids were determined, and sequence analysis showed that none of the hydrophilic segments of these proteins were labelled (Bisson et al, 1982;Hoppe et al, 1983b).…”