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

HPLW, a designed VEGF (Vascular Endothelium Growth Factor) receptor-binding peptide, assumes a well folded ß-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 ß-turn formation, whose COi-HNi + 3 hydrogen bond (Asp10 → Arg7) appears particularly stable. At 316 K, once the ß-turn is completely formed, the two ß-strands pair, very likely starting by Trp5 and Trp13, which thus play a key role also in the final step of the ß-hairpin folding. Overall, here we describe a multi-state hierarchical folding pathway of a highly structured ß-hairpin, which can be classified as a broken-zipper mechanism.

Functional binding surface of a ß-hairpin VEGF receptor targeting peptide determined by NMR spectroscopy in living cells.

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.

Design, structural and biological characterization of a VEGF inhibitor ß-hairpin-constrained peptide.

The design, structural and biological characterization of a novel VEGF inhibitor peptide is described. The peptide was designed on the ß5-ß6 hairpin region of Placenta Growth Factor. NMR studies showed that the peptide assumes in solution a ß-hairpin conformation similarly to the corresponding region of the natural ligand. In vitro experiments on endothelial cells demonstrated that the peptide is able to inhibit VEGF biological activity. This molecule represents a novel molecular entity to modulate VEGF activity and with potential application in therapy and diagnosis of angiogenesis-dependent diseases.

Semi-synthesis of labeled proteins for spectroscopic applications.

Since the introduction of SPPS by Merrifield in the 60s, peptide chemists have considered the possibility of preparing large proteins. The introduction of native chemical ligation in the 90s and then of expressed protein ligation have opened the way to the preparation of synthetic proteins without size limitations. This review focuses on semi-synthetic strategies useful to prepare proteins decorated with spectroscopic probes, like fluorescent labels and stable isotopes, and their biophysical applications. We show that expressed protein ligation, combining the advantages of organic chemistry with the easy and size limitless recombinant protein expression, is an excellent strategy for the chemical synthesis of labeled proteins, enabling a single protein to be functionalized at one or even more distinct positions with different probes.