Quantitative analysis of membrane protein–amphiphile interactions using resonance energy transfer

VALERIA LEVI; JUAN PABLO F.C. ROSSI; PABLO R. CASTELLO; F. LUIS GONZÁLEZ-FLECHA

ANALYTICAL BIOCHEMISTRY

Elsevier

Año: 2003 vol. 317 p. 171 - 179

0003-2697

emplate for Electronic Submission to ACS Journals <!-- @page { size: 21.59cm 27.94cm; margin: 2cm } P { margin-bottom: 0.21cm } --> This work describes a simple method to determine the association constant of amphiphiles to membrane proteins. The method uses a fluorescent phospholipid probe, which senses the competition among unlabeled amphiphiles for positions on the transmembrane surface of the protein. The contact between the probe and the protein surface is detected through resonance energy transfer. We have analyzed theoretically this process deriving a general equation for the dependence of the energy transfer efficiency on the composition of the micelles/bilayers in which the protein is inserted. This equation includes an exchange constant Kex,i, for each amphiphile, which gives a measure of its affinity for the protein with respect to that of one set as the reference. We applied this method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.