Although much has been learned about the design of models of β-sheets during the last decade, modest fold stabilities in water and terminal fraying remain a feature of most β-hairpin peptides. In the case of hairpin capping, nature did not provide guidance for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally applicable, "unnatural" beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a net contribution of 6 þ kJ∕mol to β-hairpin stability, surpassing all other interactions that stabilize β-hairpins including the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel β-strands and reduces fraying at the ends of hairpins and other β-sheet models. Utilizing this motif, 10-to 22-residue peptide scaffolds of defined stereochemistry that are greater than 98% folded in water have been prepared. The β-cap can also be used to staple together short antiparallel β-strands connected by a long flexible loop.beta sheets | capping stabilization | peptide hairpins | Trp/Trp interactions C apping motifs are well-known features of protein and peptide secondary structure; specifically, terminal alpha helix caps (especially N caps) are common both in proteins and designed peptides (1-4). The basis for helix capping is straightforward: countering the helix macrodipole and providing additional H-bonding interactions (1). Caps increase the fold population of isolated α-helical peptides and have been a boon to the design of α-helix models. β-Structures have capping requirements wholly different from those of helices; the ends of canonical β-sheets and hairpins do not have dipole moments or unsatisfied H bonds.The a priori design of model β-sheet systems (5), focused on hairpins, has lagged behind that of α-helix counterparts. The key discoveries that improved β-hairpin stabilities outside of protein contexts have been sequences with good turn propensities, for example D-Pro-Gly (pG) (6), heterochiral pP (7), and Aib-Gly (8) [or less favorably, Asn-Gly (NG) (5, 9)] and the incorporation of optimized cross-strand pairings [most notably Trp/Trp pairs flanking the turn (10-14)]. However, longer hairpin models are typically still frayed at the termini; to date, fully folded spectroscopic reference values have only been attained via cyclization (15-17). With the exception of cyclization, mutations at terminal sites have yielded only modest changes in hairpin stability; terminal coulombic effects (ΔΔG U ¼ 1.5-2.5 kJ∕mol) standing as the only generally observed capping effect (11,18,19). Pi-cation interactions have also been shown to provide significant hairpin stabilization (20), but instances in which the interaction appears near the ends of hairpins have provided only marginal stability increases (18,(21)(22)(23)). An unnatural π-cation interaction has also been shown to stabilize the turn in a tripeptide (24). There has been limited evidence for hairpin fold sta...

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i -2 Trp ring, a particularly stable (ΔG U > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH 2 , results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed "edge-to-face" Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater β-propensity of Trp. The β-propensity of Trp is context dependent; but, for the systems studied, always greater than that of Thr (by 0.4 -4.7 kJ/mol). At non-H-bonded positions remote from the turn, two alternative edgeto-face geometries are observed and there is no evidence of additional stabilization due to the Trp/ Trp interaction. The NMR structuring shift diagnostics of edge-to-face Trp/Trp, Trp/Lys π-cation, and Trp/Gly-H N interactions have been defined. The latter can give rise to > 3 ppm upfield shifts for the Gly-H N in -WX n G-units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred "face" residue in aryl/aryl pairings, presumably due to its greater π basicity.

Mutational optimization of two long-range interactions first observed in Ac-WINGKWT-NH 2 -a) bifurcated H-bonding involving the threonine amide H N and sidechain OH and the N-terminal acetyl carbonyl and b) an H-bond between the entgegen-H N of the C-terminal amide and the indole ring of Trp6 which stabilizes a face-to-edge indole/indole interaction between Trp1 and Trp6 -has afforded ≤10 residue systems that yield a remarkably stable fold in water. Optimization was achieved by designing a hydrophobic cluster that sequesters these H-bonds from solvent exposure. The structures and extent of amide H/D exchange protection for CH 3 CH 2 CO-WIpGXWTGPS (p = D-Pro, X = Leu or Ile) were determined. These two systems are greater than 94% folded at 298K (97.5% at 280K) with melting temperatures > 75 °C. The fold appears to display minimal fluxionality; a well converged NMR structure rationalizes all of the large structuring shifts observed and we suggest that these designed constructs can be viewed as microproteins.

The quantum yield of tryptophan (Trp) fluorescence was measured in 30 designed miniproteins (17 β-hairpins and 13 Trp-cage peptides), each containing a single Trp residue. Measurements were made in D2O and H2O to distinguish between fluorescence quenching mechanisms involving electron and proton transfer in the hairpin peptides, and at two temperatures to check for effects of partial unfolding of the Trp-cage peptides. The extent of folding of all the peptides also was measured by NMR. The fluorescence yields ranged from 0.01 in some of the Trp-cage peptides to 0.27 in some hairpins. Fluorescence quenching was found to occur by electron transfer from the excited indole ring of the Trp to a backbone amide group or the protonated side chain of a nearby histidine, glutamate, aspartate, tyrosine or cysteine residue. Ionized tyrosine side chains quenched strongly by resonance energy transfer or electron transfer to the excited indole ring. Hybrid classical/quantum mechanical molecular dynamics simulations were performed by a method that optimized induced electric dipoles separately for the ground and excited states in multiple π–π* and charge-transfer (CT) excitations. Twenty 0.5-ns trajectories in the tryptophan's lowest excited singlet π–π* state were run for each peptide, beginning by projections from trajectories in the ground state. Fluorescence quenching was correlated with the availability of a CT or exciton state that was strongly coupled to the π–π* state and that matched or fell below the π–π* state in energy. The fluorescence yields predicted by summing the calculated rates of charge and energy transfer are in good accord with the measured yields.

Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9–7.5 μs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.