Endothelin receptor in virus-like particles: ligand binding observed by fluorescence fluctuation spectroscopy

Biochemistry
Lenka ZemanováRalf Heilker

Abstract

The functional analysis of transmembrane receptor proteins is frequently hampered by the difficulty to produce sufficiently homogeneous receptor preparations that preserve the physiological biomembrane integration of the receptor protein. To improve the receptor protein density in the lipid bilayer and to maintain the physiological lipid-protein environment, a novel method has been established that enables the selective integration of transmembrane receptors into a virus-like particle (VLiP). Here we have studied the binding of tetramethylrhodamine-labeled endothelin-1 (TMR-ET-1) to VLiP-integrated endothelin A receptor (ET(A)R) by fluorescence fluctuation spectroscopy. The concentration of TMR-ET-1 was determined by fluorescence correlation spectroscopy (FCS). These measurements also confirmed that the free ligand is monomeric in solution in our experiments. Fluorescence intensity distribution analysis (FIDA) was used to quantify the fraction of ligands bound to ET(A)Rs in the VLiPs. For the interaction between ET-1 and VLiP-integrated ET(A)Rs, K(D) values of 0.5 nM and 0.3 nM were determined from ligand and receptor titration experiments, respectively. For comparison, a FIDA analysis was also carried out with ET(A)Rs in membr...Continue Reading

Citations

Dec 11, 2007·Biophysical Journal·Bin WuJoachim D Müller
Apr 9, 2008·Biophysical Journal·Jody L Swift, David T Cramb
Aug 30, 2005·Current Opinion in Chemical Biology·Richard J Middleton, Barrie Kellam
Sep 24, 2013·Biochimica Et Biophysica Acta·Rajashri SridharanMark E Dumont
Oct 20, 2005·Journal of Biomolecular Screening·Wayne R LeifertEdward J McMurchie
Jun 24, 2008·Journal of Biomolecular Screening·Jessi Wildeson JonesMartha P Brown
Jul 9, 2008·Langmuir : the ACS Journal of Surfaces and Colloids·Jong Ho HanQuan Cheng

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