Investigations of protein-lipid interactions in model membranes: Influence of aromatic anchoring residues and buried polar residues

To investigate in detail the interactions between transmembrane proteins and the lipid bilayers in which they are constituted, designed model peptides with selective isotopic labels were synthesized and analyzed by means of solid-state deuterium NMR spectroscopy. Starting from the well-characterized model peptide GWALP23, acetyl-GGALW(LA)6LWLAGA-amide, several Trp to Tyr mutations were compared to evaluate their respective interfacial anchoring abilities. It was found that Tyr, substituted on either or both termini, can effectively anchor the transmembrane α-helix, which then adopts a similar transmembrane topology in a range of bilayer thicknesses. Nevertheless, a consistent ~10° shift in tilt direction (helix rotation) is observed when a Tyr is substituted for Trp and found to be terminal-dependent (i.e. in opposite direction on each end). The fluorescence emission spectra from the single remaining Trp residue in YGWALP23 and YGWALP23 indicate that W is buried more deeply in the bilayer than is W. Using YGWALP23 as a host, the influence of Lys was examined at positions 12 and 14 in various lipid bilayer thicknesses. YGWALP23-K14 incorporates well into both thin and thick bilayers. It influences the peptide’s orientation by increasing the tilt magnitude (4-9°) and altering the tilt direction (60-95°). In contrast, the L12K mutant yields multiple low-intensity peaks in H NMR spectra, recorded in DOPC, indicative of multi-state behavior. Nevertheless, the peptide orients well and adopts a large tilt angle (30°) in the thinner bilayers of DLPC. YGWALP23-K12 in DOPC is observed to titrate at high pH to a neutral form that is well aligned in an orientation that is very similar to that of the host peptide without lysine. Titration of YGWALP23-K14 reveals a pKa of 6.2 in DOPC at 50 °C and different transmembrane orientations when the peptides charged lysine, neutral lysine or no lysine are compared. When Glu is added adjacent to a lysine, significantly improved NMR spectra and a stable transmembrane orientation are observed for YGWALP23-(K, E). YGWALP23-K14 experiences a small change in orientation with Glu-15 addition, but interestingly titrates much like the peptide with K14 alone. Placement of a Glu residue (E13) near R12 also improves spectra quality, especially at higher pH. It appears a stabilizing ion-pair may in some instances “rescue” the K12 or R12 peptide from its multi-state behavior in DOPC. The individual ionization states of paired ionizable residues have yet to be determined. This dissertation is approved for recommendation to the Graduate Council. Dissertation Director: _______________________________________ Dr. Roger E. Koeppe II Dissertation Committee: _______________________________________ Dr. Paul Adams _______________________________________ Dr. Dan Davis _______________________________________ Dr. Suresh Kumar Krishnaswamy Thallapuranam _______________________________________ Dr. David Paul DISSERTATION DUPLICATION RELEASE I hereby authorize the University of Arkansas Libraries to duplicate this dissertation when needed for research and/or scholarship. Agreed __________________________________________ Nicholas J. Gleason Refused __________________________________________