Long range Trp-Trp interaction initiates the folding pathway of a pro-angiogenic beta-hairpin peptide

HPLW, a designed VEGF (Vascular Endothelium Growth Factor) receptor-binding peptide, assumes a well folded beta-hairpin conformation in water and is able to induce angiogenesis in vivo. In this study, we investigated at atomic resolution the thermal folding/unfolding pathway of HPLW by means of an original multi-technique approach combining DSC, NMR, MD and mutagenesis analyses. In particular, careful NMR investigation of the single proton melting temperatures together with DSC analysis accurately delineate the peptide folding mechanism, which is corroborated by computational folding/unfolding simulations. The HPLW folding process consists of two main events, which are successive but do not superimpose. The first folding step initiates at 320 K upon the hydrophobic collapse of the Trp5 and Trp13 side-chains which stabilizes the concurrent beta-turn formation, whose COi-HNi +3 hydrogen bond (Asp10 -> Arg7) appears particularly stable. At 316K, once the beta-turn is completely formed, the two beta-strands pair, very likely starting by Trp5 and Trp13, which thus play a key role also in the final step of the beta-hairpin folding. Overall, here we describe a multi-state hierarchical folding pathway of a highly structured beta-hairpin, which can be classified as a broken-zipper mechanism.