title: Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy.
abstract: Fluorescence fluctuation spectroscopy provides information about protein interactions in the intercellular environment from naturally occurring equilibrium fluctuations. We determine the molecular brightness of fluorescent proteins from the fluctuations by analyzing the photon counting histogram (PCH) or its moments and demonstrate the use of molecular brightness in probing the oligomerization state of proteins. We report fluorescence fluctuation measurements of enhanced GFP (EGFP) in cells up to concentrations of 10 microM by using an improved PCH theory. The molecular brightness of EGFP is constant in the concentration range studied. The brightness of a tandem EGFP construct, which carries two fluorophores, increases by a factor of two compared with EGFP alone, demonstrating the sensitivity of molecular brightness as a probe for protein complex formation. Oligomerization of nuclear receptors plays a crucial role in the regulation of gene expression. We probe the oligomerization state of the testicular receptor 4 and the ligand-binding domains of retinoid X receptor and retinoic acid receptor by observing molecular brightness changes as a function of protein concentration. The large concentration range accessible by experiment allows us to perform titration experiments on EGFP fusion proteins. An increase in the molecular brightness with protein concentration indicates the formation of homocomplexes. We observe the formation of homodimers of retinoid X receptor ligand binding domain upon addition of ligand. Resolving protein interactions in a cell is an important step in understanding cellular function on a molecular level. Brightness analysis promises to develop into an important tool for determining protein complex formation in cells.
authors.affiliation: School of Physics and Astronomy, University of Minnesota, 116 Church Street Southeast, Minneapolis, MN 55455, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Wei
authors.forename: Joachim D
authors.initials: JD
authors.lastname: Müller
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D049452:Green Fluorescent Proteins; D007834:Lasers; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry*; D008962:Models, Theoretical; D018168:Receptors, Retinoic Acid / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D047488:Retinoid X Receptors; D013050:Spectrometry, Fluorescence / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry; D014162:Transfection
chemical_list: D008024:Ligands; D008164:Luminescent Proteins; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D049452:Green Fluorescent Proteins