Nuclear magnetic resonance and circular dichroism (CD) studies of isolated peptides corresponding to WT and mutant OmpA signal sequences are reported; all of the peptides adopt substantial amounts of alpha-helical structure both in 1:1 (v/v) trifluoroethanol (TFE)/water and in sodium dodecyl sulfate (SDS) micelles. In TFE/water, the helix begins after the positively charged N-terminal residues and is most stable in the hydrophobic core, which correlates with results obtained previously for other signal sequences. The helix is weaker between the hydrophobic core and the C-terminus; such a break in the helix appears to be common to other signal peptides studied previously and could be of functional importance. No clear correlation could be established between the helicity of the peptides in TFE/water and their in vivo activities. All the peptides have a higher alpha-helix content in SDS than in TFE/water, and there is a good correlation between helix content in SDS and in vivo activity. Helicity in SDS for the functional peptides increases both at the N-terminus and in the hydrophobic core, and is driven by a strong association of the core with the hydrophobic chains of the detergent. The extension of the helix toward the N-terminus may be a result of neutralization of the N-terminal positive charges by the headgroups of the micelles, which removes unfavorable electrostatic interactions with the helix dipole. All these comparisons were facilitated by the use of upfield shifts of H alpha protons in helical regions relative to random coil chemical shifts, which also yielded estimates of helical content that correlated well with the CD results.

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.

Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.

The higher order structure of antibacterial polymyxin B (PxB), an N-acylated pentacationic (4−10)-cyclic decapeptide, is determined from NMR data by simulated annealing calculations. The antibacterial selectivity of PxB against Gram-negative organisms suggests that PxB must participate in specific microscopic interactions with these organisms, and the structure of PxB provides insights into these interactions. Significance of the topological flexibility of certain parts of the structure in relation to the membrane-mimetic environment is developed to suggest the presence of two distinct and specific phosphoester binding sites per PxB. Although disordered in water, PxB remains in a monomeric form and adopts a well-defined structure in aqueous trifluoroethanol (TFE). Circular dichroism results show a comparable structure in aqueous TFE and on anionic vesicles. Docking and energy minimization calculations show that the two phosphoester binding sites are essentially on the same face of the structure. The topology of the ring is locked in a fixed relationship between residues 6, 7, and 10. However, the pucker of the ring changes residues 4 and 5 on one side and residues 8 and 9 on the other side. The structural flexibility within the NMR constraints permits occupancy of the sites individually or simultaneously, in a 10 to 14 Å range for the phosphorus-to-phosphorus distance between the two sites. Thus, for interactions at the Gram-negative cell surface, a PxB molecule could not only bind to the headgroup of one or two phosphatidylglycerols, but remarkably, the two sites could also simultaneously accommodate the 1,4‘-diphosphodiglucosamine of lipid A backbone of a lipopolysaccharide. The observed combination of both fixed and flexible regions of PxB, referred to as higher order structure, accounts for its ability to perform a range of microscopically distinct functions guided by the local environment at the bacterial cell surface.