title: Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell.
abstract: Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins.
authors.affiliation: Laboratory of Molecular Cell Dynamics, Graduate School of Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Oura
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
authors.forename: Johtaro
authors.initials: J
authors.lastname: Yamamoto
authors.affiliation: Laboratory of Molecular Cell Dynamics, Graduate School of Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
authors.forename: Hideto
authors.initials: H
authors.lastname: Ishikawa
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Mikuni
authors.affiliation: Laboratory of Molecular Cell Dynamics, Graduate School of Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
authors.forename: Ryousuke
authors.initials: R
authors.lastname: Fukushima
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D057446:Hydrodynamics; D056004:Molecular Dynamics Simulation; D009010:Monte Carlo Method; D066329:Protein Aggregates; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D066329:Protein Aggregates; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins