Structural Determinants of the Unusual Helix Stability of a De Novo Engineered Vascular Endothelial Growth Factor (VEGF) Mimicking Peptide

Abstract

Understanding how an amino acid sequence folds into a well organized three-dimensional structure remains a challenge. The interest in protein folding comes from the possibility to predict the protein structure from genome-derived sequence, design proteins with new fold and understand protein misfolding. Peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. Many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of nonnatural amino acids or introducing constraints. In this paper, we report on the stability characterization, using CD, NMR and MD studies, of a designed, a-helical, 15-mer peptide (named QK), composed only of natural amino acids (sequence AcKLTWQELYQLKYKGI-NH2), which activates the VEGFdependent angiogenic response. The QK peptide shows an unusual thermal stability, whose structural determinants have been determined. These results could have implication in the field of protein folding and in the design of helical structured scaffolds for the realization of peptides for applications in chemical biology. As recently described, the NMR structure of QK in pure water presents a central helical sequence (residues 4–12), which corresponds to the VEGF N-terminal helix (residues 17–25), flanked by Nand C-capping regions. The helical conformation of QK represents an important prerequisite for its biological activity, since the isolated peptide, corresponding to the helix region of VEGF, does not assume a helical conformation and does not have significant biological activity. Interestingly, QK represents one of the very few examples of bioactive helical designed peptides, composed of only natural amino acids. To gain an insight into the molecular determinants of QK helical propensity, we examined the effect of the temperature on the QK structure through NMR and CD analyses. Primarily, the aggregation state of the peptide under conditions identical to those used in the NMR structure determination was confirmed by NMR DOSY experiments (see Supporting Information). The DOSY-derived diffusion coefficient value of 1.98@10 10 ms 1 is consistent with a QK monomer state. QK structure variations upon temperature increase were followed by TOCSY experiments. In the 298– 343 K range only small changes of the backbone chemical shifts were observed (Table 1 Supporting Information). The temperature dependences of Ha chemical shift deviations from the random coil values (DdHa) are reported in Figure 1a. Unusually, the chemical shift index (CSI) analysis indicates that at 343 K the peptide retains at least the 80% of the helix conformation at 298 K and the slight reduction occurs uniformly in 4–12 region (Figure 1a). The thermal behavior was also analyzed by CD spectroscopy which allowed [a] D. Diana, Prof. Dr. R. Fattorusso Dipartimento di Scienze Ambientali, Seconda UniversitC di Napoli via Vivaldi 43, 81100 Caserta (Italy) Fax: (+39)0823-274605 E-mail : roberto.fattorusso@unina2.it [b] B. Ziaco, Prof. Dr. C. Pedone, Dr. L. D. DIAndrea Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16 80134 Napoli (Italy) Fax: (+39)081-2534574 E-mail : ldandrea@unina.it [c] Dr. G. Colombo, Dr. G. Scarabelli Istituto di Chimica del Riconoscimento Molecolare, CNR via Bianco, 9, 20131 Milano (Italy) [d] Dr. A. Romanelli Dipartimento delle Scienze Biologiche UniversitC di Napoli “Federico II” via Mezzocannone 16, 80134 Napoli (Italy) Supporting information for this article is available on the WWW under http://www.chemistry.org or from the author: Peptide synthesis, circular dichroism, nmr spectroscopy and molecular dynamic simulations.