Application of laser fluorimetry for determiningthe influence of a single amino-acid substitutionon the individual photophysical parametersof a fluorescent form of a fluorescent protein mRFP1

Abstract

Individual photophysical parameters of thechromophore of a fluorescent protein mRFP1 and its twomutants (amino-acid substitution at position 66 – mRFP1/Q66C and mRFP1/Q66S proteins) are determined. For thispurpose, apart from conventional methods of fluorimetry andspectrophotometry, nonlinear laser fluorimetry is used. It isshown that the individual extinction coefficients of thechromophore of proteins correlate (correlation coefficientabove 0.9) with the volume of the substituted amino-acidresidue at position 66 (similar to the positions of theabsorption, fluorescence excitation and emission maxima).