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.
Highlights
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
We found the possibility that the orientation of each enhanced green fluorescent protein (EGFP) in EGFP tandem-oligomers can be analyzed using pol-Fluorescence correlation spectroscopy (FCS)
Our newly developed polarization-dependent fluorescence correlation spectroscopy (pol-FCS) instrument (Fig. 1a) and the global fitting of relaxation time of the triplet state allow the observation of rotational diffusion properties of EGFPs with a clear dependence on solution viscosity
Summary
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. Fluorescence correlation spectroscopy (FCS) allows the study of molecular dynamics in solutions and cells[1,2,3] while changing the translational diffusion properties of the molecule. We simultaneously obtain short and long distance spatial information This means that pol-FCS can simultaneously provide the molecular diffusion properties in nano- and microenvironments using rotational and translational diffusions, respectively. The pol-FCS has the potential to study the diffusional barrier structure in the surrounding environment by comparing the rotational diffusion in nanoenvironment and the translational diffusion in the microenvironment. We found the possibility that the orientation of each EGFP in EGFP tandem-oligomers can be analyzed using pol-FCS
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