title: Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell.
abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K(d), for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K(d) values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Manisha
authors.initials: M
authors.lastname: Tiwari
authors.forename: Shintaro
authors.initials: S
authors.lastname: Mikuni
authors.forename: Hideki
authors.initials: H
authors.lastname: Muto
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins; D046912:Multiprotein Complexes / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D052002:NF-kappa B p50 Subunit; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D000097573:Red Fluorescent Protein; C489548:NFKB1 protein, human; C498143:RELA protein, human; C485184:enhanced green fluorescent protein
keywords: Dissociation constant; EGFP; FCCS; FCS/FCCS; Fluorescence measurement; Heterodimer; IPT; K(d); LSM; Live cell imaging; NFkB; NFκB; NLS; dissociation constant; enhanced green fluorescent protein; fluorescence cross-correlation spectroscopy; immunoglobulin-like plexin transcription factor; laser scanning microscopy; mCherry tandem dimer; mCherry(2); nuclear factor kappa B; nuclear localization signal; p50/p65