| Chapter title |
Measurement of Transmembrane Peptide Interactions in Liposomes Using Förster Resonance Energy Transfer (FRET).
|
|---|---|
| Chapter number | 2 |
| Book title |
Membrane Proteins
|
| Published in |
Methods in molecular biology, January 2013
|
| DOI | 10.1007/978-1-62703-583-5_2 |
| Pubmed ID | |
| Book ISBNs |
978-1-62703-582-8, 978-1-62703-583-5
|
| Authors |
Ambalika Khadria, Alessandro Senes |
| Abstract |
Present day understanding of the thermodynamic properties of integral membrane proteins (IMPs) lags behind that of water-soluble proteins due to difficulties in mimicking the physiological environment of the IMPs in order to obtain a reversible folded system. Despite such challenges faced in studying these systems, significant progress has been made in the study of the oligomerization of single span transmembrane helices. One of the primary methods available to characterize these systems is based on Förster resonance energy transfer (FRET). FRET is a widely used spectroscopic tool that provides proximity data that can be fitted to obtain the energetics of a system. Here we discuss various technical aspects related to the application of FRET to study transmembrane peptide oligomerization in liposomes. The analysis is based on FRET efficiency relative to the concentration of the peptides in the bilayer (peptide:lipid ratio). Some important parameters that will be discussed include labeling efficiency, sample homogeneity, and equilibration. Furthermore, data analysis has to be performed keeping in mind random colocalization of donors and acceptors in liposome vesicles. |
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