Membrane-active peptides (MAPs) are short-length peptides whose function can be affected through interactions with membrane surfaces. In the case of the acidity-triggered rational membrane (ATRAM) peptide, it possesses high solubility in solution, with the ability to insert into membranes under acidic conditions. However, the molecular details of ATRAM’s mechanism, in particular its behavior in solution as it transitions to the membrane surface, remain unclear. We hypothesize that multiple repeated segments of nonpolar residues in ATRAM facilitate the organization of higher-order oligomers in solution, which subsequently leads to insertion in a multimeric state. In this study, we carried out hydrogen-deuterium exchange (HDX) mass spectrometry experiments combined with molecular dynamics (MD) simulations to characterize the conformational changes of ATRAM and its two variants, Y-ATRAM and K2-ATRAM, as a function of peptide composition. Our HDX experiments utilize our in-house vibrating sharp spray ionization (VSSI) technique, which has been shown to facilitate excellent mixing of sample streams and to retain the solution-state structure of proteins while in the gas phase during MS data acquisition. MD simulations were used to screen ATRAM and its variants under conditions that discern differences in oligomerization in solution. In our HDX experiments, we detect three different charge states which exhibit different amounts of HDX. The most prevalent state is the [M+3H]<sup>3+ </sup>state, with 42%, 13 %, and 39% deuterium uptake for ATRAM, Y-ATRAM, and K2-ATRAM, respectively. In our MD simulations, we only observe a formation of stable oligomers for systems with a higher number of monomers (<i>N</i> = 6, 8), indicating that ATRAM is either a monomer or higher-order aggregate in solution. Our combined experimental and computational approach is the first step in generating a fundamental framework for understanding the relationship between sequence, conformational sampling, and behavior in the aqueous and membrane phases of membrane-active peptides such as ATRAM, which will allow for intelligent design of future ATRAM variants with enhanced targeting properties.

Understanding the conformation in solution of membrane-active peptides using molecular dynamics simulations and HDX-MS

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