title: Detection of tryptophan to tryptophan energy transfer in proteins.
abstract: Förster resonance energy transfer (FRET) studies usually involve observation of intensity or lifetime changes in the donor or acceptor molecule and usually these donor and acceptor molecules differ (heterotransfer). The use of polarization to monitor FRET is far less common, although it was one of the first methods utilized. In 1960, Weber demonstrated that homotransfer between tryptophan molecules contributes to depolarization. He also discovered that the efficiency of homotransfer becomes much less effective upon excitation near the red-edge of the absorption. This "red-edge effect" was shown to be a general phenomenon of homotransfer. We have utilized Weber's red-edge effect to study tryptophan homotransfer in proteins. Specifically, we determined the polarization of the tryptophan fluorescence upon excitation at 295 nm and 310 nm (near the red-edge). Rotational diffusion leads to depolarization of the emission excited at either 295 nm or 310 nm, but homotransfer only contributes to depolarization upon excitation at 295 nm. Hence, the 310/295 polarization ratio gives an indication of tryptophan to tryptophan energy transfer. In single tryptophan systems, the 310/295 ratios are generally below 2 whereas in multi-tryptophan systems, the 310/295 ratios can be greater than 3.
authors.affiliation: Department of Cell and Molecular Biology, University of Hawaii at Manoa, 1960 East-West Road, Honolulu, Hawaii 96822, USA.
authors.forename: Pierre D J
authors.initials: PD
authors.lastname: Moens
authors.forename: Michael K
authors.initials: MK
authors.lastname: Helms
authors.forename: David M
authors.initials: DM
authors.lastname: Jameson
mesh_terms: D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D005454:Fluorescence Polarization; D006801:Humans; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D011506:Proteins; D014364:Tryptophan