Folding Propensity and Biological Activity of Peptides: The Effect of a Single Stereochemical Isomerization on the Conformational Properties of Bombinins in Aqueous Solution Argante Bozzi, 1 Maria Luisa Mangoni, 2 Andrea C. Rinaldi, 3 Giuseppina Mignogna, 2 Massimiliano Aschi 4 1 Dipartimento di Scienze e Tecnologie Biomediche, Universita ` L’Aquila e Consorzlo INBB, Italy 2 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Universita ` di Roma ‘La Sapienza’, Roma, Italy 3 Dipartimento di Scienze e Tecnologie Biomediche, Universita ` di Cagliari, Cagliari, Italy 4 Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita ` de L’Aquila, L’Aquila, Italy Received 18 March 2008; revised 16 April 2008; accepted 16 April 2008 Published online 5 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21006 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com INTRODUCTION C ationic antimicrobial peptides (AMPs) are effector molecules of the ancient host defense system against microbial infections, known as innate immunity. 1,2 They are produced by all living organisms and in contrast with the clonal acquired immunity, which is present only in higher vertebrates, AMPs represent a fast and energy-effective mechanism that initially plays a key role in counteracting invading pathogens. 3–7 Despite differences in their size and secondary structure, most AMPs display a net Folding Propensity and Biological Activity of Peptides: The Effect of a Single Stereochemical Isomerization on the Conformational Properties of Bombinins in Aqueous Solution Correspondence to: Massimiliano Aschi; e-mail: aschi@caspur.it ABSTRACT: Folding propensities of bombinins H2 and H4, two members of amphibian bombinins H, a family of 17–20 residue a-helical peptides, have been investigated by means of circular dichroism (CD) measurements and molecular dynamics (MD) simulations. The two peptides, with primary structure IIGPVLGLVGSALGGLLKKI- NH2 and differing only for the configuration of the second aminoacid (an L-isoleucine in H2 and a D-alloisoleucine in H4) behave rather differently in solution. In particular both CD measurements and MD simulations indicate that bombinin H2 shows a markedly higher tendency to fold. From a careful inspection of MD trajectories it emerges that the stereochemical isomerization mutation of residue 2 to D-alloisoleucine in H4 peptide, drastically decreases its ability to form intrapeptide contacts. MD simulations also indicate that the conformational sampling in both systems derives from a subtle combination of energetic and entropic effects both involving the peptide itself and the solvent. The present results have been finally paralleled with preliminary information on bombinins H2 and H4 biological activity, i.e. interaction with membrane, supporting the hypothesis of an ‘‘already folded’’ conformation in water rather than interfacial folding tenet. # 2008 Wiley Periodicals, Inc. Biopolymers 89: 769–778, 2008. Keywords: antimicrobial peptides; frog skin; circular dichroism; molecular dynamics (MD); free energy Contract grant sponsor: Italian Ministry of Education, University and Research Contract grant numbers: PRIN 2004, 2005 V V C 2008 Wiley Periodicals, Inc. Biopolymers Volume 89 / Number 9 769