ABSTRACT
A yellow fluorescent protein (YFP) present in a strain of bioluminescent bacteria is shown here to not only modify the color and intensity of the emission, as already known and attributed to the interaction of YFP with a luciferase intermediate, but remarkably to confer a negative temperature dependence to the in vitro system. The in vitro bioluminescence decay rate is actually independent of temperature in the range 5-25° C, at ~ 1 mM YFP concentration. Several hypotheses are considered to explain this effect, based either on inactivation of YFP itself at higher temperature, or on its binding equilibrium with the luciferase intermediate. The first hypothesis is favored. Fluorescence anisotropy measurements show that YFP loses its chromophore at higher temperature, but this alone cannot account for the negative temperature dependence. Gel chromatography shows the existence of an inactive YFP dimer, and the formation of more dimer at higher temperatures cannot be ruled out, but is unlikely in our experimental conditions. Conformational changes may contribute to YFP inactivation. To our knowledge there is no prior example of an enzymatic reaction in which the rate is slower at higher temperature, within a physiological range.