NMR structures and molecular dynamics simulation of hylin-a1 peptide analogs interacting with micelles Edson Crusca Jr., a Amanda Souza Câmara, b Carolina Oliveira Matos, c Reinaldo Marchetto, a Eduardo Maffud Cilli, a Luciano Morais Lião c and Aline Lima de Oliveira c,d * Antimicrobial peptides are recognized candidates with pharmaceutical potential against epidemic emerging multi-drug resistant bacteria. In this study, we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to determine the unknown structure and evaluate the interaction with dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles with three W 6 -Hylin-a1 analogs antimicrobial peptides (HyAc, HyK, and HyD). The HyAc, HyK, and HyD bound to DPC micelles are all formed by a unique α-helix structure. Moreover, all peptides reach the DPC micelles’ core, which thus suggests that the N-terminal modifications do not influence the interaction with zwiterionic surfaces. On the other hand, only HyAc and HyK peptides are able to penetrate the SDS micelle core while HyD remains always at its surface. The stability of the α-helical structure, after peptide-membrane interaction, can also be important to the second step of peptide insertion into the membrane hydrophobic core during permeabilization. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Keywords: W 6 -Hylin-a1 analogs; NMR; antimicrobial peptides; molecular dynamics simulations; DPC; SDS Introduction The indiscriminate use of broad-spectrum antibiotics at both hospital and community environments has led to a progressive increase in the number of multi-drug resistant strains [1–4]. Despite efforts of the scientific community to understand the underlying mechanisms of bacterial resistance to antibiotics, the development and the use of new drugs have become mandatory to stop dissemination of super-bacteria [5,6]. In this scenario, antimicrobial peptides (AMPs) have emerged as a powerful alternative for therapeutic applications, including the treatment of a broad-spectrum of pathogens, like bacteria, fungi, parasites, or enveloped viruses, with limited chance to develop resistance. Basically, the AMPs exhibit the ability to interact with biological membranes by promoting pore formation and cell lysis, which is the very first action of antimicrobial activity [7,8]. The granular glands of anurans stand for a prosperous source of AMPs in nature. In 2009, Castro et al. [9] isolated the peptide named hylin-a1 (Hy-a1, IFGAILPLALGALKNLIK-NH 2 ) from the arboreal South American frog Hypsiboas albopunctatus. This peptide exhibits antibacterial and antifungal properties, as well as cytolytic activity against human erythrocytes. Circular dichroism studies showed that Hy-a1 is unstructured in solution and requires a polar-nonpolar interface to adopt its preferential membrane-active structure [9]. Nuclear magnetic resonance (NMR) experiments of Hy-a1 in sodium dodecylsulphate (SDS) micelles confirmed that the peptide adopts a helix structure, which stays parallel to the micelle interface [10]. Permeabilization studies also showed that Hy-a1 presents different affinity to membranes composed by zwitterionic or anionic lipids, which thus suggests that electrostatic forces are associated with Hy-a1 peptide interaction on the membrane [10]. Additionally, the importance of different charges at the N-terminus of W 6 -Hy-a1 analog (IFGAIWPLALGALKNLIK-NH 2 ) in its activity was previously evaluated [11]. It has been shown that modification of the N-terminus by addition of a Lys, Asp, or even an acetyl group perturbates the amphipathic α-helix and affects its ability to interact with the cell membrane [11]. Based on these findings and the unexplored influence of structural features on the biological behavior of Hy-a1, our study focuses on determining the unknown 3D structure of Hy-a1 analogs and evaluates their interaction with dodecylphosphatidylcholine (DPC) and SDS micelles, aiming at providing more information regarding the mechanism of antimicrobial action. The 3D structures of three Hy-a1 analogs, namely HyAc: Ac-IFGAIWPLALGALKNLIK- NH 2 , HyK: KIFGAIWPLALGALKNLIK-NH 2 and HyD: DIFGAIWP LALGALKNLIK-NH 2 ), are determined by NMR spectroscopy. The molecular dynamics (MD) simulation assessed the events of * Correspondence to: Aline Lima de Oliveira, Institute of Chemistry, University of Brasília, 70910-000, Brasília, DF, Brazil. E-mail: aline.alo@gmail.com a São Paulo State University (UNESP), Institute of Chemistry, Araraquara, SP, Brazil b Institute of Physics of São Carlos, University of São Paulo, São Carlos, SP, Brazil c Institute of Chemistry, Federal University of Goiás, Goiânia, GO, Brazil d Institute of Chemistry, University of Brasília, Brasília, DF, Brazil J. Pept. Sci. 2017 Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 22 November 2016 Revised: 28 February 2017 Accepted: 13 March 2017 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/psc.3002