Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus.

The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.

Purification and characterization of minor brain proteolipids: use of fast atom bombardment-mass spectrometry for peptide sequencing.

A combination of lipophilic gel permeation chromatography and ion-exchange chromatography in organic solvents was used to purify low molecular weight proteolipids from bovine brain. Cleavage peptides were purified by HPLC and studied mainly by the fast atom bombardment--mass spectrometry technique. A proteolipid of Mr 14 000 contains several peptides from the first 113 amino acids of the major myelin proteolipid (MMPL) plus an extra unknown blocked N-terminal peptide. A proteolipid of Mr 16 000 contains smaller peptides belonging to a C-terminal fragment of MMPL of about 160 residues. These two proteolipids do not seem to be artifacts from MMPL.

[Structural data on the myelin proteolipid of apparent molecular weight 20 kDa (DM-20)]. / Données structurales sur le protéolipide de la myéline de masse moléculaire apparente 20 kDa (DM-20).

By a combination of chromatographic methods, we have obtained in an apparently homogeneous state the 24 kDa "major myelin proteolipid" (MMPL) and the 20 kDa "myelin proteolipid" (DM-20). Contrary to a commonly held view, the second one is not a conformationally different form of its major companion, as it differs markedly by its amino-acid composition, its electrophoretic behaviour after performic acid oxidation, and the results of tryptic digestion; however, they are obviously very closely related, as shown by the selective cleavages at the level of methionines and tryptophanes. These results are most simply interpreted by a single deletion in DM-20 of the hydrophilic fragment 100-140 of the (known) structure of the 24 kDa proteolipid. Lees' hypothesis of a deletion of fragment 197-267 cannot be retained.

Intrinsic fluorescence of a non-myelin apoproteolipid and evidence for the existence of conformational flexibility.

An extremely hydrophobic protein (Mr = 16000), which in its native form is only soluble in organic solvents and which differs from the myelin proteolipid (Mr = 24000), was purified to homogeneity. Intrinsic fluorescence studies on this apoproteolipid have revealed a large conformational flexibility. In the water-soluble form the emitting residues appear to be buried in a hydrophobic core while in organic solvents they are exposed to the external medium. Structural changes depending on the organic solvent are also observed. The emission characteristics of reconstituted proteoliposomes may be due to the formation of a membrane-linked complex between several proteolipid monomers.

Brain proteolipids. Isolation, purification and effect on ionic permeability of membranes.

Proteolipid apoproteins have been isolated from a whole bovine brain homogenate by chloroform/methanol extraction, and fractionated by chromatography on modified (lipophilic) Sephadex, followed by ion-exchange chromatography on CM-Trisacryl. The various final, highly hydrophobic, fractions are homogeneous (sodium dodecyl sulfate/polyacrylamide gel electrophoresis). Transmembrane ion transfers were studied by 22Na + flux and electrical conductance measurements. Single channel events were observed at low protein concentrations, in particular with one of the final homogeneous apoproteolipids of molecular mass 24 kDa.