EGFP oligomers as natural fluorescence and hydrodynamic standards.

摘要: EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion coefficients D of EGFP1–4 were determined by analytical ultracentrifugation with detection correlation spectroscopy (FCS), yielding 83.4…48.2 μm2/s 97.3…54.8 μm2/s from monomer to tetramer. A “barrels standing in a row” model agreed best the sedimentation data. Oligomerization red-shifted emission spectra without any shift absorption. Fluorescence anisotropy decreased, indicating homoFRET between subunits. lifetime decreased only slightly (4%) insignificant quenching FRET subunits non-emitting states. FCS-measured D, particle number molecular brightness depended dark states light-induced processes distinct subunits, resulting dependence illumination power different monomers oligomers. Since may be “on” (bright) or “off” (dark) states, FCS-determined apparent is not proportional that monomer. From its probability state subunit was 96% at pH 8 77% 6.38, i.e., protonation increases state. These properties oligomeric can assist interpreting results oligomerized fusion proteins biological interest.