“Snorkeling” of the Charged Sidechain of a Transmembrane Peptide as Directly Observed by Double Electron-Electron Resonance Experiment

While hydrophobic amino acids constitute the bulk of transmembrane protein domains, polar and even charged amino acids are not uncommon and often play significant roles in membrane protein function. Positioning a polar residue within the bilayer core is highly unfavorable thermodynamically; however, the free energy penalty could be minimized by “stretching” the side chain of the amino acid to bring the charged moiety closer to the bilayer surface while keeping the rest of the side chain inside the hydrophobic core. This biophysical phenomenon is known as “snorkeling”. Here we report experimental observations of “snorkeling” for nitroxide-modified side-chains upon protonation, its dependence upon the location along the transmembrane peptide helix, and how this snorkeling is affected by the membrane electrostatic surface potential. pH sensitive spin labels, either IMTSL or IKMTSL (JPCB 2009, 113, 3453) were attached to two cysteine residues positioned equidistant from the center of the WALP peptide so that the primary sequence of each peptide is palindromic, thus, ensuring symmetric location of the labels with respect to the bilayer. The change in protonation states of the nitroxide was directly observed from EPR spectra. The distance between two nitroxide moieties was measured by Q-band double electron-electron resonance (DEER) experiment. Upon protonation, the distance between the two IMTSL probes increased compared to that of the neutral forms, by approximately 3 A indicating displacements of the charged nitroxide sidechain towards the polar head region. The “snorkeling” of the label was observed to be depth dependent - no changes in the positioning of the sidechain upon protonation was observed for labels located within 10-8 A from the center of the bilayer. Supported by NSF-0843632 to TIS.