Combinatorial approaches: a new tool to search for highly structured beta-hairpin peptides

Abstract

Here we present a combinatorial approach to evolve a stable beta-hairpin fold in a linear peptide. Starting with a de novo-designed linear peptide that shows a beta-hairpin structure population of around 30%, we selected four positions to build up a combinatorial library of 20(4) sequences. Deconvolution of the library using circular dichroism reduced such a sequence complexity to 36 defined sequences. Circular dichroism and NMR of these peptides resulted in the identification of two linear 14-aa-long peptides that in plain buffered solutions showed a percentage of beta-hairpin structure higher than 70%. Our results show how combinatorial approaches can be used to obtain highly structured peptide sequences that could be used as templates in which functionality can be introduced.

Figure 1

Structure-based screening of the four separate sublibraries. Each bar represents the ratio θ₂₁₇/θ₂₀₂ obtained from the CD spectra of each peptide mixture in buffer (empty bars) and in 40% of MeOH (solid bars) solutions, with the x axis representing the defined amino acid (O_B position). For the selection of the most suitable amino acids for each position, we calculate the average θ₂₁₇/θ₂₀₂ ratio value, in aqueous and MeOH solution, for the four positions (the average values are within the error for the four positions when considered independent). Any residue whose θ₂₁₇/θ₂₀₂ value was equal (with an error of ± 0.1) or higher than the sum of the mean plus one SD, in water and/or in MeOH, was considered to favor β-hairpin formation.

Figure 2

(A) CD spectra in buffer of the 20 different peptide mixtures that define the sublibrary RG-KO_−B3TX-NG-XTXE-GR. Note the presence of an isodichroic point at 208 nm. (B and C) CD spectra of selected peptides that fold into β-hairpin structures. The CD spectra of peptides MBH12 (solid line) and MBH36 (dotted line) (sequences listed in Table 2) were acquired at 5°C in 5 mM acetate buffer (pH 5) (B) and in the presence of 40% MeOH (C). The CD spectra of the original scaffold peptide BHKE (dashed line) are included as reference.

Figure 3

¹³C_α conformational shift profile of (A) MBH12 (black bars; Aa⁹ is I) and MBH18 (gray bars; Aa⁹ is N) and (B) MBH36 (black bars; Aa⁹ is I) and MBH35 (gray bars; Aa⁹ is N). Random-coil reference values have been taken from Wishart et al. (29). The profiles are consistent with the intended β-hairpin conformation. There are some anomalous values (positive or null) that are likely reflecting those residues that do not adopt an extended conformation. This finding is consistent with the angles measured from regular β-hairpins of this size that have been found on the Protein Data Bank.

Figure 4

Observed conformational CαH chemical shift increments (Δδ_CαH) relative to the chemical shifts of the unfolded state for peptides MBH12 (black bars; Aa⁴ is W) and MBH36 (gray bars; Aa⁴ is Y).

Figure 5

Schematic diagram of the structure of peptides MBH12 and MBH36 in solution. The observed NOEs in water are shown as thick lines.

Figure 6

(A) Backbone traces of the 10 best structures obtained from NMR restraints for peptide MBH12 using DYANA. (B) Minimized mean of the 10 best NMR structures calculated for peptide MBH12. View of the hydrophobic (a) and polar face (b) of MBH12. Side chains are shown in space-fill representation.