Heterogeneous Dielectric and Hydrogen Bonding Environment of Transmembrane Peptides

A complex set of hydrogen bonding and hydrophobic interactions between the protein side chains and cellular membrane components is primary responsible for such important biophysical processes as initial protein binding/docking to cellular membranes, membrane insertion, folding, and the final adaptation of the correct transmembrane position. Although general concepts of membrane protein folding and thermodynamic stability are beginning to emerge, the experimental data on the transmembrane profile of the effective dielectric constant and the local hydrogen bond network formed by membrane protein side chains remain severely limited. Here we describe the use of an arsenal of modern spin-labeling EPR methods to profile heterogeneous dielectric and hydrogen bonding environment along a series of the alpha-helical chain of the alanine-rich WALP peptide that adopts a transmembrane orientation. Firstly, we have employed a recently described pH-sensitive cysteine-specific spin-label IMSTL (methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester) to label a series of WALP cysteine mutants. EPR titrations of such peptides reconstituted into anionic lipid bilayers yield the magnitude of relative changes in the effective dielectric constant across the bilayer in the vicinity of the peptide alpha-helix. Secondly, perdeuterated and 15N-substituted nitroxides in combination with High Field EPR at 130 GHz (D-band) were used to assess local polarity and formation of hydrogen bonds for the same series of spin-labeled WALP mutants. Finally, the nature of the hydrogen bonds observed by EPR was ascertained by a series of HYSCORE X-band measurements. It was concluded that such combination of EPR techniques significantly expands the capabilities of spin-labeling methods in studies of membrane proteins as demonstrated by deriving profiles of heterogeneous dielectric and hydrogen bonding environment along a typical transmembrane alpha-helix. Supported by NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.