The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.

The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.The purple membrane of a number of extremely halophilic bacteria-e.g., Halobacterium halobium-functions as a light-driven proton pump (1, 2). It contains a single protein, bacteriorhodopsin (Mr 26,000) with one molecule of retinaldehyde covalently bound to a lysine residue (3, 4). Bacteriorhodopsin forms a continuum of seven a helices, each of which spans the membrane and is largely embedded in it (5). Knowledge of the primary structure of bacteriorhodopsin is a prerequisite to studies of the mechanism of the proton pump and of the biogenesis of this interesting protein. In a recent paper, we reported (6) on the sequence of the first 102 amino acids and of 39 amino acids from the COOH terminus of this membrane protein. The present paper reports on its complete amino acid sequence (Fig. 1). Although no experimental details have appeared, the complete sequence has also been deduced by Ovchinnikov and coworkers (7,8), and partial sequences have been reported by other laboratories (3, 9, 10). The present sequence differs from that reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and amino acid assignments at positions 105, 111, 117, 146, and 206. Of the total 248 amino acids present in bacteriorhodopsin, 70% are hydrophobic, and there is significant clustering of the hydrophobic as well as of the hydrophilic amino acids. MATERIALS AND METHODSMaterials. N-Bromosuccinimide (NBS), fluorescamine, and isothiocyanatophenylthiocarbamoylaminopropyl (IPTAP) glass were purchased from Pierce, and trypsin treated with L-(tosylamido-2-phenyl)ethyl chloromethyl ketone, clostripain, and elastase were from Worthington Biochemicals. ['4CISuc-cinic anhydride was from New England Nuclear. Tetraethyltetraamino (TETA) and aminoethylaminopropyl (AEAP) glass were gifts from Mark Horn of Sequemat, Watertown, MA. Other materials were as described previously.Preparation of Chymotryptic and CNBr Fragments. Purple membrane was isolated from H. halobium and apomembrane was prepared as described (6). Digestion of the apomembrane with chymotrypsin gave two fragments, C-1 and C-2, which were collected by centrifugation and separated on a Sephadex LH-60 column ...

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The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y. A., Abdulaev, N. G., Feigina, M. Y., Kiselev, A. V. & Lobanov, N. A. (1977) FEBS Lett. 84,[1][2][3][4]. Chymotryptic cleavage of bacteriorhodopsin roduced two fragments, C-1 (Mr 19,000) and C-2 (M, 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.The purple membranes of a number of extremely halophilic bacteria-e.g., Halobacterium halobium-function as lightdriven proton pumps (1, 2). They contain a single protein, bacteriorhodopsin (Mr 26,000) with one molecule of retinaldehyde covalently bound to a lysine residue (3, 4). Bacteriorhodopsin forms a continuum of seven a helixes, each of which spans the membrane and is largely embedded in it (5). Knowledge of the primary structure of bacteriorhodopsin is a prerequisite to an understanding of the mechanism of proton pumping; in this paper we report on the elucidation of the sequence of the first 102 amino acids as well as-of 39 amino acids at the COOH terminus of this integral membrane protein.Partial amino acid sequences of bacteriorhodopsin have been reported (3, 6), and, more recently, extensive sequence work has been reported by Ovchinnikov and coworkers (7), although no experimental details have appeared so far.Sequence work with membrane proteins is still in its infancy, and progress has been reported only in a relatively few cases (8-10). The straightforward application of current methods for sequencing water-soluble proteins to large hydrophobic membrane proteins such as bacteriorhodopsin is not feasible. Therefore, a major aim of the present work has been the development of methods for sequencing integral membrane proteins. This included gel permeation chromatography with nonaqueous solvents, reverse-phase high-pressure liquid chromatography (HPLC) for separation of the fragments, and a well-balanced combination of gas chromatographic mass spectrometry (GCMS) and automated Edman degradation for sequencing. MATERIALS AND METHODSMaterials. The enzymes used were obtained commercially. Sequencer reagents were purchased from Pierce, LiAl2H4 and B22H6 from Alfa-Ventron (Danvers, MA), Sephadex LH-20 and LH-60 from Pharmacia, and ,uBondapak C18 columns from Waters Associates (Milford, MA).Electrophoresis of Polypeptide Fragments. Fragmenta...

The possible interaction of a second lysine with the retinylidene Schiff base of bacteriorhodopsin (Lewis, A., Marcus, M. A., Ehrenberg, B. & Crespi, H. (1978) Proc. Nati AcaSL Sci USA 75 4642-4646) has been investigated by specific incorporation of IN into the E-amino groups of the lysine residues. Comparison of resonance Raman spectra of bacteriorho. dopsin grown on 100%, 0%, and 50% labeled lysine demonstrates that '5N isotope effects on the Schiff base vibration can be accounted for by 15N labeling only at the Schiff base nitrogen. Our data also provide in situ confirmation of the linkage of the retinal chromophore with the E-amino nitrogen of lysine. Understanding the molecular mechanism by which bacteriorhodopsin in the purple membrane of Halobacterium halobium functions as a light-transducing proton pump remains a key goal in membrane research (for a review, see ref. 1). One important area under active investigation is the nature of the interaction between the retinylidene chromophore of bacteriorhodopsin and the protein, bacterioopsin (2, 3). It is believed that this interaction is responsible for the red shift of the absorption maximum of the major visible band-of bacteriorhodopsin in its lightadapted state (bR570) relative to that of the retinal chromophore (Amaj = 380 nm) and protonated retinylidene Schiff base model compounds (Am. = 440 nm) (3). Because protonation of the Schiff base can only account partially for the red shift of bR570, several types of secondary interactions have been postulated (2-5).Lewis et aL (6) have proposed a model for proton pumping by bacteriorhodopsin that involves as a key element the direct interaction of a nitrogen-containing residue, most likely lysine, with the Schiff base of bR570. This model is based partially on the evidence from resonance Raman spectroscopy that complete '5N-labeling of bacteriorhodopsin lowers the protonated Schiff base vibration (C-NEH stretch) from. 1642 (7) to 1627 cm-, whereas the deprotonated Schiff base vibration (C=N stretch) of the intermediate M412 is only shifted from 1619 (7) to 1614 cm-1. It is implied that the labeling of a non-Schiff base nitrogen contributes to the 15-cm-l isotopic shift in bR570 but does not contribute to the 5-cm-' shift in M412. Further, pHdependent kinetic effects indicate that this nitrogen-containing group is likely to be lysine (6). Although this lysine interaction model is of interest, it is not clear how an isotopic substitution of a nonchromophoric atom could directly affect a chromophore vibration such as the C=NeH stretch. For this reason, we have attempted to test the hypothesis that the labeling of a nitrogen other than the Schiff base nitrogen in bacteriorhodopsin affects the Schiff base vibration. MATERIALS AND METHODSHalobacterium halobium R1 was grown in a synthetic medium similar to that of Gochnauer and Kushner (8): 430 mg of DLalanine, 400 mg of L-arginine, 50 mg of L-cysteine, 1.3 g of Lglutamic acid, 60 mg of glycine, 440 mg of DL-isoleucine, 800 mg of L-leucine, 42.5mg of L-lysine,...

Complete amino acid sequences have been established for 19 muscle-related proteins and these proteins are each sufficiently abundant to suggest that their mRNA levels are about 0.4% or higher. Based on these considerations, a simple theoretical analysis shows that clones for most of these proteins can be identified within a complementary DNA library by sequencing cDNA inserts from 150-200 randomly selected clones. This procedure should not only rigorously identify specific clones, but it could also uncover amino acid sequence variants of major muscle proteins such as the troponins. We have determined sequences for about 20,000 nucleotides within 178 randomly selected clones of a rabbit muscle cDNA library, and report here that in addition to finding sequences encoding the two known skeletal muscle isotypes of troponin C, we have discovered sequences encoding two forms of troponin T. Over the region of nucleotide sequence overlap in the troponin T clones, the new isotype diverges significantly from its counterpart. Altogether, clones for 13 of the 19 known muscle-specific proteins were identified, in addition to the clone for the new troponin T isotype.

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trifluoroethyl-0-trimethylsilyl polyamino alcohols, the derivatives of choice for the sequencing of polypeptides by gas chromatographic mass spectrometry, has been optimized. The imphvements have minimized undesirable side reactions and resulted in a five-to tenfold increase in sensitivity over previous methods employing either lithium aluminum deuteride or hexadeuterodiborane as the reducing agent. For derivatives of certain very polar peptides increases in yield have exceeded 100-fold. The procedure has been evaluated with mixtures of synthetic oligopeptides and used in the course of the determination of the amino acid sequence of the membrane protein bacteriorhodopsin, as well as other proteins. Protein Specific enzymatic or chemical cleavage Partial hydrolysis with acid or enzymes i 1 Primary degradation fragment Complex mixture of di-to hexapeptides 3 N HCl/MeOH or CHzNz t CFsC02CHs t TEA t B2D6/THF or LiAID,/GLYME TMSDEAIPyridine Complex mixture of 0-TMS polyamino alcohols Scheme 1 @ Heyden