Quantitative analysis of membrane protein-amphiphile interactions using resonance energy transfer.
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.
Levi
Valeria
V
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.
Rossi
Juan P F C
JP
Castello
Pablo R
PR
González Flecha
F Luis
FL
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
D008051:Lipid Bilayers; D008055:Lipids; D008565:Membrane Proteins; D008823:Micelles; D010743:Phospholipids; D011721:Pyrenes; C030984:pyrene; D000252:Calcium-Transporting ATPases