Probing Dielectric and Hydrogen Bonding Gradients in Biological Membranes

Abstract

Nitroxide spin-labeling in combination with EPR spectroscopy has found many applications in studying structure and dynamics of proteins and biological membranes. Recently, there has been a substantial interest in utilizing EPR to characterize local effects of polarity and hydrogen bonding in proteins and biological membrane systems. Here we report on employing an arsenal of advanced spin-labeling EPR methods to profile heterogeneous dielectric and hydrogen bonding environment along the α-helical chain of an alanine-rich WALP peptide that is anchored in a lipid bilayer in a transmembrane orientation. A series of WALP cysteine mutants was labeled with a pH-sensitive nitroxide IMSTL (S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) that is similar in molecular volume to phenylalanine. The protonation state of this nitroxide could be directly observed by EPR allowing us to follow proton gradient across the membrane in the vicinity of the WALP α-helix, and, thus, to reconstruct the gradient in the effective dielectric constant. These experiments were complemented by assessing local polarity from characteristic changes in EPR spectra that were enhanced by the use of perdeuterated and 15N-substituted nitroxides and high field EPR at 130 GHz (D-band). Formation of hydrogen bonds between the nitroxides and membrane-penetrating water molecules was observed directly in HYSCORE X-band experiments. Such measurements allowed us to derive experimental profiles of heterogeneous dielectric and hydrogen bonding environment along a typical transmembrane α-helix. Supported by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.