The conformational properties of protein fragments have been widely studied as models of the earliest initiation events in protein folding. While native-like alpha-helices and beta-turns have been identified, less is known about the factors that underly beta-sheet formation, in particular beta-hairpins, where considerably greater long-range order is required. The N-terminal 20 residue sequence of native ferredoxin I (from the blue-green alga Aphanothece sacrum) forms a beta-hairpin in the native structure and has been studied in isolation by NMR and CD spectroscopy. Local native-like interactions alone are unable to stabilize significantly a folded conformation of the 20-residue fragment in purely aqueous solution. However, we show that the addition of low levels of organic co-solvents promotes formation of native-like beta-hairpin structure. The results suggest an intrinsic propensity of the peptide to form a native-like beta-hairpin structure, and that the organic co-solvent acts in lieu of the stabilizing influence of tertiary interactions (probably hydrophobic contacts) which occur in the folding of the complete ferredoxin sequence. The structure of the isolated hairpin, including the native-like register of interstrand hydrogen bonding interactions, appears to be determined entirely by the amino acid sequence. The solvent conditions employed have enabled this intrinsic property to be established.

The N-terminal 17 residues of ubiquitin have been shown by 1 H NMR to fold autonomously into a b-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly b-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for b-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the b-region of conformational space even in the absence of the hairpin structure.

The formation of the N-terminal b-hairpin of ubiquitin is thought to be an early event in the folding of this small protein.Previously, we have shown that a peptide corresponding to residues 1-17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U~1-17!T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U~1-17!T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 b-bulge, as in the full-length protein. The reason for the greater stability of the U~1-17!T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys11, which may indicate that a charge-charge interaction between these residues is responsible.Keywords: b-hairpin; b-sheet; NMR; peptide; peptide conformations; structure; ubiquitin; X-PLOR The b-hairpin is one of the most commonly occurring structural motifs in globular protein folds: most antiparallel b-sheets contain at least some strand pairs that are contiguous in sequence and connected by a relatively tight turn. There has also been much speculation and some experimental evidence to support the idea that hairpins may play a key role in initiating the assembly of b-structures in folding~Ptitsyn, 1991; Muñoz et al., 1997!. In recent years, there has been considerable interest in using b-hairpins as model systems for developing our understanding of b-structure and several examples both of protein fragments and of de novo designed peptides capable of folding autonomously as hairpins have now been identified~Blanco et al., 1994;Searle et al., 1995;Ramirez-Alvarado et al., 1996!. The lessons learned from exploring the effects of sequence variations in these systems have been reviewed recently~Griffiths- Jones et al., 1998;Ramirez-Alvarado et al., 1999!. Sequence variations cannot only alter the stability of hairpin structures, but also the register of the b-strands. In the arms of the hairpin, a high b-propensity of individual residues is important for stability. However, specific pairwise contacts, especially between side chains interacting across the hairpin, are also important and optimization of these is hypothesized to be an important contribution in fixing the strand register~Smith & Regan, 1995;Wouters & Curmi, 1995;Hutchinson et al., 1998!. The turn sequence is also important: it is clear from analysis of hairpins within intact protein structures that some turn types are prevalent in b-hairpins. Sequence compatibility with these preferred structures has been shown in peptide studies to be an important determinant of structural specificity and stability~Ramirez- Alvarado et al., 1997;De Alba et al., 1999;Griffiths-Jones et al., 19...

From a consideration of the interactions between non-covalent bonds, it is concluded that positively cooperative binding will occur with a benefit in enthalpy and a cost in entropy, and that negatively cooperative binding will occur with a cost in enthalpy and a benefit in entropy; experimental data support these conclusions.

A wide range of dimerisation constants (Kdim ca. 101–106 M−1) for various glycopeptide antibiotics have been determined. We consider these dimerisation constants in the light of the published X‐ray structures of the antibiotics, in particular, the relationship between Kdim and the length of a specified distance at the dimer interface. In the crystals, we find that this distance is smaller for strongly dimerising antibiotics and larger for weakly dimerising antibiotics. Thus, the dimerisation constant is correlated with tightness at the dimer interface. Despite the crystal‐packing forces exerted between adjacent dimer molecules in the crystals, the noncovalent bond distances at the dimer interface are correlated with the distances in solution (inferred from solution NMR data). These observations can account for the benefits in enthalpy, and costs in entropy, associated with positively cooperative binding.