title: Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching.
abstract: Accurate determination of the depth of membrane penetration of a fluorescent probe, attached to a lipid, protein, or other macromolecule of interest, using depth-dependent quenching methodology is complicated by thermal motion in the lipid bilayer. Here, we suggest that a combination of steady-state and time-resolved measurements can be used to generate a static quenching profile that reduces the contribution from transverse diffusion occurring during the excited-state lifetime. This procedure results in narrower quenching profiles, compared with those obtained by traditional measurements, and thus improves precision in determination of the underlying depth distribution of the probe.
authors.affiliation: Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kyrychenko
authors.affiliation: Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA. Electronic address: aladokhin@kumc.edu.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes
keywords: Bilayer penetration; Distribution analysis; Lipid-attached quenchers; Static and dynamic fluorescence quenching