Thermodynamic origin of α-helix stabilization by side-chain cross-links in a small protein

Abstract

Peptide cross-linking has been widely explored as a means of constraining short sequences into stable folded conformations, most commonly α-helices. The prevailing hypothesis for the origin of helix stabilization is an entropic effect resulting from backbone pre-organization; however, obtaining direct evidence bearing on this hypothesis is challenging. Here, we compare the folding thermodynamics of a small helix-rich protein domain and analogues containing one of three common cross-linking motifs. Analysis of the folding free energy landscapes of linear vs. cyclized species reveal consistent trends in the effect of cyclization on folding energetics, as well as subtle differences based on the chemistry of the cross link. Stabilization in all three systems arises entirely from a reduction in the entropic penalty of folding that more than compensates for an enthalpic destabilization of the folded state.

Long-range PEG stapling: macrocyclization for increased protein conformational stability and resistance to proteolysis

“…23 For example, lactam staples can be prepared via conventional peptide coupling chemistry [24][25][26][27][28][29] or by diverse chemoselective strategies, including the Ugi reaction; 30,31 direct thioester aminolysis; 32 native chemical ligation; [33][34][35] KAHA ligation; 36 traceless Staudinger ligation; 37 and a variety of enzymatic methods. [38][39][40] Other creative stapling strategies include C-H activation; [41][42][43] the Petasis reaction; 44 the Glaser reaction; 45 oxime 46,47 or hydrazone 48 formation; the copper(I)catalyzed azide-alkyne cycloaddition (CuAAC); [49][50][51][52][53][54][55] and olefin metathesis. [56][57][58][59][60][61] We are interested in understanding the origin and determinants of protein stabilization via macrocyclization/stapling in diverse structural contexts.…”

1,2,3-Triazoles as Biomimetics in Peptide Science

“…As mentioned above, a peptidomimetic with a triazole moiety has shown the structural stability of a coiled coil peptide. In addition, cyclization side-chain to side-chain by the copper-catalyzed dipolar cycloaddition in a VHP (villin headpiece) peptide shows this very well [ 68 ]. A VHP peptide has 36 residues that are stable in aqueous solution, with a compact tertiary folded structure composed of three α-helices.…”

Heteroaryl Rings in Peptide Macrocycles

“…The folding thermodynamics of several bridged peptides 1114a−c derived from a helix-rich sequence of the villin headpiece (HVP) domain was evaluated (Figure 81). 421 The analysis of the folding free energy revealed only minute differences based on the chemistry of the cross-link. Backbone preorganization was the dominant factor, regardless of the cross-linking chemistry employed.…”