Monitoring Protein Association with a Membrane Bilayer Using Ultraviolet Resonance Raman (UVRR) Spectroscopy

Abstract

Ultraviolet Resonance Raman (UVRR) as a means of studying proteins’ three states: in solution (state I), embedded on the membrane surface(state II), or inserted within the lipid bilayer (state III). pHLIP (pH low insertion peptide) is the model used for membrane proteins since it can change states based on pH going above or below 8. An artificial lipid membrane made of DMPC is made. After calculating an efficient peptide to lipid membrane ratio for insertion to use and making a suitable solution for an adequate UVRR spectrum, we have seen that when pHLIP is in state II it is partially in the membrane and gaining a helical structure. Using UVRR to study transmembrane proteins in relation to pH gives researchers insight on the functionality of many diseases. Previous studies have shown that the C helix of bateriorhodopsin, commonly referred to as pH low insertion peptide (pHLIP), is disordered in aqueous solutions and in the presence of membrane bilayers at neutral pH. At low pH, in the presence of membrane bilayers, the peptide spontaneously folds in to an alpha-helix and inserts into the membrane bilayer. Ultraviolet Resonance Raman (UVRR) has been employed to study the three states of pHLIP: in solution (State I), embedded on the membrane surface (State II), or inserted within the lipid bilayer (State III). Ideal peptide-to-lipid membrane ratios were determined using tryptophan fluorescence and ultracentrifugation was used to concentrate liposomes for UVRR measurement. UVRR studies reveal that each state is spectroscopically distinct. The amide S mode, a marker for non-helical structure disappears upon going from neutral to low pH.