title: Application of three-photon excitation FCS to the study of protein oligomerization.
abstract: Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log-log plot of intensity against laser power.
authors.affiliation: Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Suman
authors.initials: S
authors.lastname: Ranjit
authors.forename: Alexander
authors.initials: A
authors.lastname: Dvornikov
authors.forename: David A
authors.initials: DA
authors.lastname: Holland
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Reinhart
authors.forename: David M
authors.initials: DM
authors.lastname: Jameson
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D007834:Lasers; D008099:Liver / Q000201:enzymology; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods; D025221:Phosphofructokinases / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D014364:Tryptophan; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases