Minimization and optimization of designed beta-hairpin folds

Abstract

Minimized beta hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, DeltaG(U)(298) >or= +7 kJ/mol) and 12-residue (KTWNPATGKWTE, DeltaG(U)(298) = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-pi interaction between Lys(1) and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hepsilon3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the -TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small DeltaC(p) for unfolding (+420 J K(-)(1)/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH(2)) with the fold stability reduced to DeltaG(U)(278) = -0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also form the same hairpin-stabilizing W/W interaction.

Figure 1

The nomenclature for positions in [2:2]/[2,4], [3:5], and [4:4]/[4:6] (Thornton classification) β hairpins; T indicates turn or loop positions, S indicates strand positions: [2:2]- and [4:4]-hairpins have an H-bond between the S+1 NH and the S−1 carbonyl. The S±even, non-hydrogen bonded (NHB), positions have their Hα atoms directed inward and display short Hα/Hα distances. The S±odd positions have their Hα’s outwardly directed with the sidechains displayed on the top surface of the hairpin as depicted here. The odd-numbered strand positions are also designated as “H-bonded pairs”.

Figure 2

CD spectra recorded for peptide HP5W4.

Figure 3

An overlay (least squares fitted over the residue 3 – 14 backbones) of representative structures from the NMR ensembles of trpzip4 and peptide HP5W4.

Figure 4

NMR and CD melts for the HP6 series of peptides.

Figure 5

Overlaid structures in the HP7 NMR ensemble, the N-terminal strand is in front of the C-terminal strand in this view.

Figure 6

Representative melting studies of a series of HP7 analogs. A. The temperature dependence of the most upfield aryl-H signal (Hε3 of W3), B. CD melts of the same series; the observed “adjusted-[θ]₂₂₇” (see Methods) values for (K1R)-HP7 and (N4D)-HP7 are multiplied by factors of 1.14 and 1.07, respectively, to raise them just above the values recorded for HP7 since all other data indicates that these analogs are at least as well-folded as the parent system. The fitted curves in panel B are fourth order polynomials, rather than based on a folding model; presumably they capture the curvature of the experimental data and can be used to obtain Tm values by the second derivative method.