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

VALERIA LEVI, JUAN P.F.C. ROSSI, PABLO R. CASTELLO, AND F. LUIS GONZ
ALEZ FLECHA

ANALYTICAL BIOCHEMISTRY

Año: 2003 vol. 31 p. 171 - 179

0003-2697

This work describes a simple method for determining the association constant of amphiphiles to membrane proteins. The methoduses a fluorescent phospholipid probe, which senses the competition among unlabeled amphiphiles for positions on the transmembranesurface of the protein. The contact between the probe and the protein surface is detected through resonance energytransfer. We have analyzed theoretically this process deriving a general equation for the dependence of the energy transfer efficiencyon the composition of the micelles/bilayers in which the protein is inserted. This equation includes an exchange constant for eachamphiphile, which gives a measure of its affinity for the protein with respect to that of an amphiphile set as the reference. We appliedthis method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.
2003 Elsevier Science (USA). All rights reserved.