Previous conformational analysis of 10-residue linear peptides enabled us to identify some cross-strand side-chain interactions that stabilize β-hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N- and C-termini of these 10-residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on β-hairpin formation, we have designed two 15-residue β-hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15-residue peptides suggest that the influence of stabilizing factors for β-hairpin formation, in particular, cross-strand side-chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in β-hairpin folding.

A previous NMR investigation of model decapeptides with identical β-strand sequences and different turn sequences demonstrated that, in these peptide systems, the turn residues played a more predominant role in defining the type of β-hairpin adopted than cross-strand side-chain interactions. This result needed to be tested in longer β-hairpin forming peptides, containing more potentially stabilizing cross-strand hydrogen bonds and side-chain interactions that might counterbalance the influence of the turn sequence. In that direction, we report here on the design and 1H NMR conformational study of three β-hairpin forming pentadecapeptides. The design consists of adding two and three residues at the N- and C-termini, respectively, of the previously studied decapeptides. One of the designed pentadecapeptides includes a potentially stabilizing R-E salt bridge to investigate the influence of this interaction on β-hairpin stability. We suggest that this peptide self-associates by forming intermolecular salt bridges. The other two pentadecapeptides behave as monomers. A conformational analysis of their 1H NMR spectra reveals that they adopt different types of β-hairpin structure despite having identical strand sequences. Hence, the β-turn sequence drives β-hairpin formation in the investigated pentadecapeptides that adopt β-hairpins that are longer than the average protein β-hairpins. These results reinforce our previous suggestion concerning the key role played by the turn sequence in directing the kind of β-hairpin formed by designed peptides.

A 12-residue peptide designed to form an α-helix and self-associate into an antiparallel 4-α-helical bundle yields a 0.9 Å crystal structure revealing unanticipated features. The structure was determined by direct phasing with the “Shake-and-Bake” program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal α-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the α-helices run antiparallel and are closely spaced (9–10 Å center-to-center). The sheets are more loosely packed against each other (13–14 Å between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-α-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7–11° rather than 18°).

Here we describe the NMR conformational study of a 20-residue linear peptide designed to fold into a monomeric three-stranded antiparallel β-sheet in aqueous solution. Experimental and statistical data on amino acid β-turn and β-sheet propensities, cross-strand side-chain interactions, solubility criteria, and our previous experience with β-hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C-NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12-residue peptides encompassing the N- and C-segments of the designed sequence indicates that the 20-residue peptide folds into the expected conformation. Assuming a two-state model, the exchange kinetics between the β-sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil-β-sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in β-sheet formation and stability.