Measuring the Bilayer Depth Dependence of the SCN Infrared Probe Group using Poly-L Transmembrane Peptides

Abstract

Most conventional protein characterization techniques cannot be applied to membrane proteins due to their large size, complicated structures, and dependence on their natural environments. Vibrational probe groups are one new method that can provide site-specific structural information with a fast intrinsic time scale in membrane proteins. The SCN group has a unique CN stretching band whose frequency reports on membrane burial. Four poly-L transmembrane peptides were synthesized with cysteines at varying positions so that the SCN vibrational probe group could be inserted at four different known burial depths. The four variants were cyanylated using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) with at least an 80% yield determined by liquid chromatography-mass spectrometry (LCMS). Size exclusion chromatography on a Superdex Peptide column was used to purify the peptides, which were then mixed with POPC lipids and inserted into rehydrated bilayers and vesicle samples via a sequence of freeze-thaw and sonication cycles. Infrared spectroscopy (IR) spectra were collected to observe the stretching frequencies and bandwidths of the inserted SCN vibrational probes.