title: Quantitative analysis of membrane protein-amphiphile interactions using resonance energy transfer.
abstract: This work describes a simple method for determining 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 for each amphiphile, which gives a measure of its affinity for the protein with respect to that of an amphiphile set as the reference. We applied this method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas, Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Buenos Aires C1113AAD, Argentina.
authors.forename: Valeria
authors.initials: V
authors.lastname: Levi
authors.forename: Juan P F C
authors.initials: JP
authors.lastname: Rossi
authors.forename: Pablo R
authors.initials: PR
authors.lastname: Castello
authors.forename: F Luis
authors.initials: FL
authors.lastname: González Flecha
mesh_terms: D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D008051:Lipid Bilayers; D008055:Lipids; D008565:Membrane Proteins; D008823:Micelles; D010743:Phospholipids; D011721:Pyrenes; C030984:pyrene; D000252:Calcium-Transporting ATPases