Investigating Peptide Assembly in a Membrane Environment

Abstract

ObjectiveThis study seeks to determine how the properties of a lipid membrane influence protein folding and assembly in model systems using hydrogen‐detuerium exchange mass spectrometry (HDX‐MS).MethodsThe folding and assembly of small model peptides in large unilamellar vesicles (LUVs) have been investigated using HDX‐MS. We currently have results for the 13‐residue, cationic peptide indolicidin in LUVs prepared with 1,2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) or 1,2‐Dioleoyl‐sn‐glycero‐3‐phosphoglycerl (DOPG).ResultsOur HDX‐MS results indicate that free indolicidin is fully deuterated at all measured time points, including the shortest (30s). Results with LUVs show differences between LUVs made of DOPC (a zwitterionic phospholipid) and DOPG (an anionic phospholipid). Indolicidin mixed with DOPC LUVs has slightly less deuterium incorporation (~3 fewer deuterium), compared to the free peptide in the absence of LUVs. Indolicidin with DOPG LUVs shows additional protection from hydrogen‐deuterium exchange (~5 fewer deuterium), compared to free indolicidin. Preliminary MS/MS experiments suggest that HDX protection occurs at the C‐terminal end of indolicidin.ConclusionsEvidence from several studies shows that MPs fold into their “correct” structures even in certain lipid‐free environments, suggesting that the membrane may not be required for proper MP folding. However, many researchers go to great lengths to mimic the physical and chemical environment of the membrane and claim that characteristics including curvature, fluidity, and symmetry are significant. Overall, we plan to investigate three model peptides known to insert into the membrane and oligomerize to form membrane‐active structures. The model peptides represent different structural classes and their individual structures have already been well‐characterized by traditional biophysical methods.Our results with free indolicidin are consistent with the fact that, in solution, indolicidin remains in a monomeric form and adopts an extended, linear structure. The results with LUVs indicate that the peptide does interact with both types of LUVs. More protection from HDX occurs with the DOPG LUVs, suggesting that the protein may be embedding itself deeper into the DOPG (anionic) LUVs, compared to DOPC (zwitterionic) LUVs. The surface charge of the model membrane may affect the peptide's ability to associate with the membrane. The MS/MS results suggest that the peptide's C‐terminus may be responsible for insertion into both LUV types. We are currently varying the model membranes to examine the effects of fluidity and curvature, as well.Support or Funding InformationThis work was supported by a Major Research Instrumentation grant (MRI1531435) from the National Science Foundation and a grant from the Murdock Charitable Trust.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.