Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D 1H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain 'random coil' amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these 'random coil' peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K., (1979) Biopolymers, 18, 285-297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH2 [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67-81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp4l within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paranyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 ,uM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.

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Myoglobin has been extensively studied as a model system for protein folding in vitro. As part of an ongoing study of myoglobin folding, we have synthesized a series of peptide fragments corresponding to portions of the sequence of the sperm whale protein. The conformational preferences of these peptides have been investigated by circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution. In this paper we describe the folding propensities of two peptides (Mb-G and Mb-H), corresponding to the G- and H-helix segments of the myoglobin sequence. The Mb-G peptide shows evidence of a very small population of helical conformations in aqueous solution, both by CD and NMR. By contrast, the monomeric Mb-H peptide is found by CD to adopt a significant population (ca. 30%) of ordered helix and by NMR to populate helical conformations in rapid dynamic equilibrium with unfolded states. The Mb-H peptide undergoes a well-characterized, concentration-dependent monomer-tetramer equilibrium. At peptide concentrations greater than 1 mM there is an increase in the population of helix, to approximately 85% according to the CD spectrum, through self-association to form a tetramer. Both medium-range NOE connectivities and a CD spectrum characteristic of ordered helix are observed at low peptide concentrations, establishing that helical conformations are present in the monomeric state of Mb-H. The relative helicity at various sites throughout the Mb-H peptide has been estimated using a novel method for assessing the distribution of helical populations based on the relative magnitudes of medium-range d alpha beta (i,i+3) NOE connectivities. The population of ordered helix is seen to be highest in the center of the peptide sequence; the ends of the peptide show evidence of pronounced fraying.

Two synthetic peptides corresponding to sequences in HIV-1LAI gp41, (aa558-595) and T20 (aa 643-678), are strong inhibitors of HIV-1 viral fusion, having EC50 values of 1 microgram/mL and 1 ng/mL, respectively. Previous work suggested that T21 forms a coiled-coil structure in PBS solution, while T20 is primarily nonhelical, and that the inhibitory action of these peptides occurs after the interaction between the viral gp120 protein and the cellular CD4 receptor [Wild, C.T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., Matthews, T. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9770 and references therein]. The current study uses sedimentation equilibrium (SE), circular dichroism (CD), and viral-fusion assays to quantitatively investigate peptide structure and peptide-peptide interactions. SE analyses of T21 (1-100 microM) indicate that the peptide self associates via a monomer/dimer/tetramer equilibrium; in addition, T20 is monomeric in the range of 1-10 microM and exhibits a complicated monomer/tetramer equilibrium between 20 and 100 microM. Singular value decomposition analyses of the CD spectra of T21 and T20 indicate that the helical content of these peptides in PBS solution is 90% and 20%, respectively. A structural interaction between the two peptides is detected by CD at several concentration ratios of T20:T21. These experiments emphasize that T20 interacts specifically with the tetrameric form of T21. Truncated forms of T20 also exhibit structural interactions with T21 at varying concentration ratios. The ability of T20 and the truncated peptides to interact structurally with tetrameric T21 correlates with antiviral activity. Implications of these findings are discussed in terms of proposed mechanisms of membrane fusion inhibition and the structural changes which occur in gp41 during membrane fusion.