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Article Cite This: ACS Omega 2017, 2, 8536−8542
Protonation Enhances the Inherent Helix-Forming Propensity of pHLIP Chitrak Gupta and Blake Mertz* C. Eugene Bennett Department of Chemistry, West Virginia University, 217 Clark Hall, Morgantown, West Virginia 26506, United States S Supporting Information *
ABSTRACT: Cell-penetrating peptides (CPPs) can be potentially used in targeted delivery of therapeutic cargoes. However, their conformation in solution is poorly understood. We employed molecular dynamics simulations to probe the structural fluctuations of an anionic CPP, pH Low Insertion Peptide (pHLIP), in solution to determine the effects of modifications to selected residues on the structure of pHLIP. Two types of modifications were tested: (1) protonation of aspartic acid residues and (2) point mutations known to affect the acid sensitivity of pHLIP. pHLIP samples conformations ranging from coil to helix to sheet, and modifications to pHLIP lead to subtle shifts in the balance between these conformations. In some instances, pHLIP is as likely to form a helical conformation as it is to form an unstructured coil. Understanding the behavior of pHLIP in solution is necessary for determining optimal conditions for administration of pHLIP and design of promising pHLIP variants.
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INTRODUCTION Cell-penetrating peptides (CPPs) are a class of molecules with potential applications ranging from antimicrobial agents to vehicles for drug delivery.1 The pH Low Insertion Peptide (pHLIP) is a fairly unique CPP, in that it is highly anionic (overall charge of −5), long (AEQNPIYWARYADWLFTPLLLLDLALLVDADEGT), and sensitive to changes in pH.2,3 In solution, pHLIP is in a coiled conformation (state I); when exposed to the cell membrane under alkaline conditions, pHLIP binds to the membrane surface, remaining in a coiled conformation (state II); upon acidification of the environment, the acidic residues in pHLIP are protonated, leading to folding into an α-helix and insertion into the membrane (state III).4 Although circular dichroism (CD) and fluorescence spectroscopy are commonly used to monitor the transition of pHLIP from state I → state II → state III, they cannot provide atomistic insights into the interactions that characterize each state. In particular, the behavior of pHLIP in solution (i.e., state I) is poorly understood. This lack of understanding is an issue, as most experiments with pHLIP require low peptide concentrations (
