comparison between the rotational correlation times of a soluble protein obtained by electron paramagnetic spin resonance and fluorescence depolarization

ABSTRACT

Electron Spin Resonance [ESR] and Fluorescence polarization were used to study the rotational correlation times of a Histidine containing protein [HPr] isolated from Escherichia coli. HPr was derivatized with 3-[maleimidomethyl] 2,2,5,5, tetra-methyl-1-pyrrolidinyloxyl [TEMPO-maleimide] as an ESR probe and [4-phenylspiro [Furan-2 [3H]1- phthalon] 3,3' dione [fluorescamine] as a fluorescent probe. Different sucrose concentrations were used to increase the viscosity of the HPr solution. Fluorescence polarization data were taken at 10 degree and 20 degree to further effect the viscosity. Experimental data reflected the expected increase in rotational correlation times by increasing the viscosity. The differences between the experimental and the calculated data are explained on the basis that the derivatised probes rotate at faster rates which were independent from those of HPr itself or due to partial denaturation of the protein