De novo design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions

Abstract

Design of helical super secondary structural motifs is expected to provide important scaffolds to incorporate functional sites, thus allowing the engineering of novel miniproteins with function. An ␣,␤-dehydrophenylalanine containing 21-residue apolar peptide was designed to mimic the helical hairpin motif by using a simple geometrical design strategy. The synthetic peptide folds into the desired structure as assessed crystallographically at 1.0-Å resolution. The two helices of the helical-hairpin motif, connected by a flexible (Gly)4 linker, are docked to each other by the concerted influence of weak interactions. The folding of the peptide without binary patterning of amino acids, disulfide bonds, or metal ions is a remarkable observation. The results demonstrate that preferred interactions among the hydrophobic residues selectively discriminate their putative partners in space, leading to the unique folding of the peptide, also a hallmark of the unique folding of hydrophobic core in globular proteins. We demonstrate here the engineering of molecules by using weak interactions pointing to their possible further exploitation in the de novo design of protein super secondary structural elements. D e novo protein design endeavors to understand the frustrating complexity of protein architecture and has the ambitious goal of constructing novel molecules with predetermined structure and function(s) (1-4). Most design strategies rely on information from the rich database of solved protein structures (5, 6). Further, the observed patterning of polar͞nonpolar residues and their different modes of interaction with the solvent is a major consideration in achieving compaction (1-4, 7). A conceptually distinct approach is to achieve stereochemical control over the local folding of the peptide by incorporation of conformation-restricting residues. For example, ␣,␤-dehydrophenylalanine (⌬Phe) or ␣-aminoisobutyric acid residues nucleate helical conformation whereas D-Pro nucleates hairpins (8-11). The success in establishing these ''folding rules'' has encouraged subsequent attempts to design and construct super secondary structures, in particular, the apparently simple helical hairpin (helix-turn-helix) motif. Many attempts in which designed hydrophobic helices were connected by linker sequences containing various coded͞noncoded amino acids with a tendency to break continuous helix formation, e.g., -aminocaprionic acid, L-lactic acid, Gly-Pro, D-Phe-Pro, and Gly-Dpg, etc., have failed to realize the desired folded conformation (9, 12). Therefore the stabilization of the monomeric helical hairpin motif without binary patterning of polar͞nonpolar residues has proved to be challenging.Our design strategy for stabilization of the helical hairpin motif is based on a shift of paradigm from overemphasis on the linker region (9) toward the optimization of weak interactions between helices (termed long-range interactions). We have used ⌬Phe, the dehydro analogue of phenylalanine, with a double bond between C ␣ and C ␤ atoms, which mak...

Synergy with Rifampin and Kanamycin Enhances Potency, Kill Kinetics, and Selectivity ofDeNovo-Designed Antimicrobial Peptides

“…⌬Fm and ⌬Fmscr (a scrambled-sequence version of ⌬Fm) (Table 1) are isomeric, monomeric decapeptides containing the nonproteinogenic amino acid ␣,␤-didehydrophenylalanine (⌬F) in their sequences. Such didehydroamino acids are well known to constrain peptides in a helical conformation (24). Previous results from our laboratory established that ⌬Fm (MIC of 135.8 g/ml [concentration of 100 M] and MBC of Ͼ611.1 g/ml [Ͼ500 M]) has poor potency and kill kinetics (no killing effects observed in 60 min even at 4ϫ MIC) against Escherichia coli ML35p (10).…”

“…Both the coupling of amino acids and the Fmoc deprotection were monitored by the Kaiser test (17). ⌬Phe was introduced into peptides as an Fmoc-X-⌬Phe azalactone (where X is Gly, Lys [Boc], or Ala) dipeptide block (24), which was allowed to couple overnight in DMF. At the completion of assembly of the peptides, following Fmoc removal, the amino termini were acetylated using 20% acetic anhydride in DCM.…”

Rationale-Based, De Novo Design of Dehydrophenylalanine-Containing Antibiotic Peptides and Systematic Modification in Sequence for Enhanced Potency

“…The presence of more than one ⌬Phe in peptides has been shown to constrain the peptide in a 3 10 or ␣-helical conformation, along with providing enhanced resistance to enzymatic degradation compared to their phenylalanine-containing counterparts (23,29). We used a de novo-designed prototype undecapeptide peptide (VS1) incorporating ⌬Phe as a lead in an optimization strategy to design three sets of peptides with the same basic motif, where ⌬Phe is placed at two residue spaces.…”

Helix packing motif common to the crystal structures of two undecapeptides containing dehydrophenylalanine residues: Implications for the de novo design of helical bundle super secondary structural modules

“…22 This self-association concept was successfully exploited by us in the design of super secondary structure helixturn-helix, where the adjacent helices that docked to each other are of opposite screw sense. 23 However, a strategy has to be evolved for the development of a discrete folded super secondary structural motif in which the interacting helices are right-handed, for which there has been until now no crystal structure containing a DPhe residue as a model for pairing and interaction. Here, we report the synthesis and characterization of two closely related undecapeptides-namely, Ac-Gly 1 10 -helices.…”