Tryptophan zippers: Stable, monomeric β-hairpins

Abstract

The design of peptides that have well-defined tertiary structures tests our understanding of the principles governing the folding of larger proteins. Designed sequences (23–28 aa) that adopt the β-β-α “zinc finger” fold have been notable successes (1–3) and are among the smallest protein domains demonstrated to fold cooperatively without assistance from metal ions or disulfide crosslinks (2, 3). Shorter peptides that form three-stranded β-sheets (4) or exhibit partial β-hairpin structure (5–10) undergo extremely broad thermal transitions, suggesting a lower limit of 20–30 aa for a stable tertiary fold. Here we report well-folded peptides of half that size: 12- and 16-residue monomeric β-hairpins, stabilized by tryptophan-tryptophan cross-strand pairs, exhibit reversible and highly cooperative thermal unfolding transitions in water. These tryptophan zippers (trp-zips) are minimal units of β tertiary structure and remarkably, for short β-hairpins, have the thermodynamic properties of typical folded proteins.

Short peptides with significant hairpin structure recently have emerged as β-sheet model systems (5, 6). However, even the best reported examples are only marginally stable (ΔG_unf ≈ 0 at 298 K) (5–10). It is not known whether this stability represents an upper limit for these very small peptides and, if it does not, what residue substitutions might further promote hairpin folding. In particular, cross-strand tertiary interactions have not yet been extensively investigated in these systems. To begin to address this, we determined an experimental energy scale for substitutions in a nonhydrogen-bonded (NHB) strand position of a disulfide-cyclized β-hairpin (11). Unexpectedly, tryptophan was much more stabilizing in this site than other amino acids (≥0.5 kcal⋅mol^{) (}11). Further studies showed that paired, cross-strand NHB residues in the cyclized hairpin made roughly independent contributions to stability; thus, a tryptophan-tryptophan cross-strand pair was highly stabilizing (and the best NHB residue pair we identified) (12). Accordingly, it seemed likely that introduction of a second tryptophan-tryptophan cross-strand pair would greatly stabilize the hairpin conformation. We thought that this additional stability would be sufficient to drive folding, even in the absence of a covalent disulfide constraint.

We find that this combination of two Trp-Trp nonhydrogen-bonded cross-strand pairs is generally useful in stabilizing β-hairpin structures. We have characterized four variants having different turn sequences and lengths. In each case, the peptides are highly water-soluble, well-structured, and monomeric. High-resolution NMR structures of three of the peptides show the two cross-strand Trp pairs interdigitating in a zipper-like motif on the surface of the folded peptide. This arrangement of the indole side chains confers unusual spectroscopic properties on the folded molecules, and folding therefore can be monitored readily by changes in CD signal. The stabilities of the trpzips are significantly higher than those reported for other small β-structures (ΔG_unf = 0.6–1.7 kcal⋅mol ^{at 298 K); on a per-residue basis, the trpzips have stabilities comparable to much larger protein domains (Δ}G_unf,max = 60–120 cal⋅mol^{⋅residue). Because of their small size, unusual stability, and very favorable spectroscopic properties, trpzip peptides should prove useful and quite simple systems in which to study β-structure and folding.}

Thermal melts were acquired with 20 μM peptide samples in 20 mM potassium phosphate, pH 7.0. σ ≡ reduced apparent molecular weight, as determined from sedimentation data fit to a nonideal single-species model (see Materials and Methods). n.d. ≡ not determined; the thermal denaturation curve of trpzip4 was identical at 5-fold higher peptide concentration (100 μM vs. 20 μM). Thermal unfolding parameters of ΔH = 11,600 cal⋅mol ^{and Δ}S = 39 cal⋅mol^{⋅K have been reported for the gb1 peptide, assuming Δ}C_p = 0 (7; see also Materials and Methods).

All peptides were synthesized as C-terminal amides; p ≡ d-proline. Residue numbers for the gb1 peptide correspond to those of the parent 56-residue B1 domain.

Resonance assignments and coupling constants for trpzip1, trpzip2, and trpzip4 are available in Tables 4–6.

Acknowledgments

Abbreviations

Footnotes