Molecular pH Probes at a Protein-Lipid Interface: Assessment of Local Dielectric Environment for Transmembrane Peptide

Abstract

Spin-labeling EPR spectroscopy has found many applications in studying structure and dynamics of proteins and biological membranes. Recently, there has been substantial interest in utilizing EPR to characterize local effects of polarity and hydrogen bonding in these systems. Here we report on employing an pH-sensitive EPR probe IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) to profile heterogeneous dielectric environments along the α-helix of a WALP peptide integrated in a lipid bilayer. A series of symmetrically positioned double cysteine mutants were labeled with a pH-sensitive nitroxide and the protonation state of IMTSL was directly observed by EPR. Q-band DEER experiments with double-labeled WALPs were employed to derive nitroxide-nitroxide distances of nitroxides before and after the protonation and, therefore, the positions of pH probes with respect to lipid bialyer. Thus, for the first time measurements of local electrostatics at peptide-bilayer interface were based on direct distance measurements rather than on assumptions on the probe location. For double-labeled WALP consecutive protonation of symmetrically positioned nitroxide tags was observed. The difference in observable pKa values was interpreted in terms of electrostatic interaction energy between titratable probes allowing us to estimate effective dielectric constant. Supported by NSF-0843632 to TIS.