Probing membrane proteins: Monitoring proton-translocation quantitatively

Abstract

Proton-translocating membrane-bound proteins create and maintain pH gradients across membranes, playing important bioenergetic and regulatory roles. Some of these proteins are implied in ageing and neurodegeneration, others in tumor biology and drug resistance. Unfortunately, membrane proteins are notoriously difficult to study. They are vastly underrepresented in the growing body of protein structures, and recent proteomics advances have mostly involved soluble proteins.We present a system for quantitatively monitoring proton-translocation by membrane proteins. The protein is reconstituted into large vesicles, which provide a well defined environment. Optionally, the protein can be first fused to a cytochrome, facilitating unidirectional reconstitution1. The pH inside and outside the vesicles is monitored simultaneously using novel membrane-impermeable nanoprobes2,3. Using CCD detection, full spectral coverage of the nanoprobes and any protein-bound chromophores is delivered at up to ms time resolution. The proteoliposomes are then subjected to computer-controlled, semi-automated titrations of substrate and/or pH changes4.Key improvements over similar methodologies previously employed are the advantageous characteristics of the nanoprobes, the precision and speed of the titration system, and the CCD detection technology. Combined, these features ensure robust, fast, quantitative results on proton-pumping stoichiometries across membranes.Using this method, we characterize the lipid bilayer of the vesicles in terms of passive ion leakage and stability. We then investigate subunits of respiratory Complex I, and their antiporter homologues, reconstituted into liposomes (see Sindra Peterson Arskold's poster).1. Gustavsson, T., Eek, M., Leiding, T., Peterson Arskold, S., and Hagerhall, C. (2008) Submitted to Biochimica et Biophysica Acta Bioenergetics.2. Finikova, O., Galkin, A., Rozhkov, V., Cordero, M., Hagerhall, C., and Vinogradov, S. (2003) J. Am. Chem. Soc. 125, 4882-4893.3. Leiding, T., Gorecki, K., Vinogradov, S., Hagerhall, C., and Peterson Arskold, S. (2008) Submitted to Analytical Biochemistry.4. Autonomous Science Machines TM.