Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching |
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Authors: | Alexander Kyrychenko Alexey S. Ladokhin |
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Affiliation: | Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA |
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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. |
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Keywords: | Static and dynamic fluorescence quenching Bilayer penetration Distribution analysis Lipid-attached quenchers |
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