title: Identifying and measuring transmembrane helix-helix interactions by FRET.
abstract: Specific interactions between helical transmembrane domains (TMs) play essential roles in the mechanisms governing the folding, stability and assembly of integral membrane proteins. Thus, it is appealing to identify helix-helix contacts and to seek the structural determinants of such interactions at the molecular level. Here, we provide a protocol for detecting and measuring specific helix-helix interactions in liposomes by Förster resonance energy transfer (FRET), using peptides corresponding to the TM domains of an integral membrane protein. We give a detailed procedure and practical guidelines on how to design, prepare, handle, and characterize fluorescently labeled TM peptides reconstituted in large unilamellar lipid vesicles. We also discuss some critical aspects of FRET measurements to ensure the correct analysis and interpretation of spectral data. Our method uses tryptophan/pyrene as the donor-acceptor FRET pair, but it can be easily adapted to other fluorescence pairs and to other membrane mimetic environments. The ability to identify crucial interhelical contacts is a valuable tool for the study of the stability, assembly, and function of the important and experimentally challenging helical membrane proteins.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
authors.forename: Damien
authors.initials: D
authors.lastname: Thévenin
authors.forename: Tzvetana
authors.initials: T
authors.lastname: Lazarova
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011721:Pyrenes / Q000737:chemistry; D013223:Statistics as Topic; D013861:Thiocyanates / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
chemical_list: D008081:Liposomes; D008565:Membrane Proteins; D010455:Peptides; D010743:Phospholipids; D011721:Pyrenes; D013861:Thiocyanates; C025244:ammonium ferrothiocyanate; D014364:Tryptophan; C030984:pyrene