In Situ FTIR ATR Spectroscopy of the Preparation of an Oriented Monomolecular Film of Porin Omp32 on an Internal Reflecting Element by Dialysis

Abstract

The development of a reproducible technique for the preparation of oriented porin Omp32 monolayers on an attenuated total reflection (ATR) internal reflection element (IRE) was the principal aim of this work. A procedure earlier applied for two-dimensional crystallization of membrane proteins [Paul, A.; Engelhardt, H.; Jakubowski, U.; Baumeister, W. Biophys. J. 1992, 61, 172−188] could be successfully adapted. Layer formation was performed in a spectroscopic dialysis cell in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) micelles by the removal of the detergent (n-octylpolyoxyethylene, OPOE). This setup enabled in situ monitoring of the monolayer formation by means of the Fourier transform infrared (FTIR) single beam sample reference ATR technique. The time course of OPOE and sodium azide extraction by dialysis and the formation of the protein/lipid layer on top of the IRE was evaluated from corresponding time-dependent absorbance changes in the IR spectra. Both OPOE and azide featured a first-order kinetics, whereas the synchronous adsorption of porin to the Ge IRE resulted in a sigmoidal time behavior. At the beginning of the dialysis, the concentration of OPOE was just above the critical micellar concentration (cmc) which is about 0.25%. As the cmc was reached by dialysis, Omp32 adsorption was accelerated, reaching saturation after about 5 h. Quantitative analysis of the Omp32/lipid layer gave strong evidence for a well-ordered monolayer with the barrel axis of the Omp32 trimer being approximately oriented perpendicular to the supporting IRE. Two to six DMPC molecules were detected per Omp32 monomer.