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This paper provides an introduction to fundamental conformational states of polypeptides in the beta-region of phi,psi space, in which the backbone is extended near to its maximal length, and to more complex architectures in which extended segments are linked by turns and loops. There are several variants on these conformations, and they comprise versatile scaffolds for presentation of side chains and backbone amides for molecular recognition and designed catalysts. In addition, the geometry of these fundamental folds can be readily mimicked in peptidomimetics., (Copyright 2005 Wiley Periodicals, Inc.)

Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.

Daptomycin is a 13-residue cyclic lipopeptide with Ca2+-dependent bactericidal activity against a variety of high-risk pathogens. Ring closure in daptomycin is via an ester linkage between the side chain of Thr4 and the C-terminal carboxyl of the main chain; the N-terminal residue is capped by a decanoyl aliphatic chain. Extensive NMR data obtained under solution conditions that minimize aggregation have provided constraints for a detailed conformational analysis of daptomycin in aqueous solution, which should facilitate the rational design of improved analogs and enhance understanding of its mode of action. Transannular and shorter-range nuclear Overhauser effects (NOEs) as well as amide temperature shifts and 3J(NH alpha) coupling constants indicate that daptomycin adopts a well-defined conformation containing a distorted hairpin formed by Gly5-D-Ala6 type II' beta-turn. A number of hydrophobic moieties (the lipid N-cap and the Trp1 and Kyn13 side chains) are clustered at one end of the hairpin, while neutral polar and anionic residues are localized on the other end, leading to amphipathicity in the molecule. These features suggest a mode of action in which the large hydrophobic cluster of the peptide interacts with the acyl chain region of a membrane. This interaction may be facilitated by Ca2+ ions, both by neutralizing the anionic charges and by favoring association with the membrane head groups. Interestingly, our findings differ from two recent articles in which the aqueous conformation of Ca2+-free daptomycin is reported to lack a well-defined conformation (D. Jung, A. Rozek, M. Okron, and R. E. W. Hancock, Chemistry & Biology, 2004, Vol. 11, pp. 949-957) or is suggested to populate an alternate conformation (L.-J. Ball, C. M. Goult, J. A. Donarski, J. Micklefield, and V. Ramesh, Organic & Biomolecular Chemistry, 2004, Vol. 2, pp. 1872-1878)., (Copyright 2005 Wiley Periodicals, Inc)

The experiments described here explore the role of local sequence in the folding of cellular retinoic acid binding protein I (CRABP I). This is a 136-residue, 10-stranded, antiparallel beta-barrel protein with seven beta-hairpins and is a member of the intracellular lipid binding protein (iLBP) family. The relative roles of local and global sequence information in governing the folding of this class of proteins are not well-understood. In question is whether the beta-turns are locally defined by short-range interactions within their sequences, and are thus able to play an active role in reducing the conformational space available to the folding chain, or whether the turns are passive, relying upon global forces to form. Short (six- and seven-residue) peptides corresponding to the seven CRABP I turns were analyzed by circular dichroism and NMR for their tendencies to take up the conformations they adopt in the context of the native protein. The results indicate that two of the peptides, encompassing turns III and IV in CRABP I, have a strong intrinsic bias to form native turns. Intriguingly, these turns are on linked hairpins in CRABP I and represent the best-conserved turns in the iLBP family. These results suggest that local sequence may play an important role in narrowing the conformational ensemble of CRABP I during folding.

A central question in protein folding is the relative importance of locally encoded structure and cooperative interactions among residues distant in sequence. We have been exploring this question in a predominantly beta-sheet protein, since beta-structure formation clearly relies on both local and global sequence information. We present evidence that a 24-residue peptide corresponding to two linked hairpins of cellular retinoic acid-binding protein I (CRABP I) adopts significant native structure in aqueous solution. Prior work from our laboratory showed that the two turns contained in this fragment (turns III and IV) had the highest tendency of any of the eight turns in this anti-parallel beta-barrel to fold into native turns. In addition, the primary sequence of these two turns is well conserved throughout the structural family to which CRABP I belongs, and residues in the turns and their associated hairpins participate in a network of conserved long-range interactions. We propose that the strong local-sequence biases within the chain segment comprising turns III and IV favor longer-range interactions that are crucial to the folding and native-state stability of CRABP I, and may play a similar role in related intracellular lipid-binding proteins (iLBPs).

We have recently shown that two of the beta-turns (III and IV) in the ten-stranded, beta-clam protein, cellular retinoic acid-binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976-7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side-chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer-range stabilizing interactions across the beta-turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a beta-bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long-range influences in the mechanism of folding of this predominantly beta-sheet protein., (Copyright 2004 Wiley Periodicals, Inc.)

In our effort to understand the structural requirements for the antimicrobial activity of cecropin A (CA) and melittin (M), we synthesized the normal, enantio, retro and retroenantio hybrid analogs; we related activity to their sequence, chirality, amide bond direction (helix dipole) and end group charges. To compare the effect of the end groups, each of these analogs was synthesized both with an acid and an amide C-terminus and also with and without an N alpha-acetyl N-terminus. The all-L- and all-D-enantiomers of several cecropin-melittin hybrids were previously found to be equally potent against several bacterial species, and no chiral effect was observed. This general rule has now been confirmed and extended. However, two exceptions have been found. All-L-CA(1-13)M(1-13) acid was 5 times and 9 times less potent than the all-D-analog, respectively, toward gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. All-L-CA(1-7)M(2-9) acid was 5 times and 14 times less active against S. aureus and P. aeruginosa, respectively, than its all-D acid isomer. The corresponding D- and L-retro analogs differed only marginally. A role for proteolytic enzymes has been implicated as a cause for these differences in the activities of L- and D-enantiomers. In all cases, blocking the alpha-amine by acetylation had no significant effect on potency. The retro and retroenantio analogs of CA(1-13)M(1-13) acid were as potent as their normal and enantio analogs against all the test bacteria. The C-terminal amides also showed similar potency against four test bacteria. It should be noted that the negative end of the helix dipole of a normal peptide points toward the C-terminus, whereas it points away in the case of a retro derivative when viewed in the direction of the normal sequence.