A bacterial luciferase reaction with a negative temperature coefficient attributable to protein-protein interaction

Abstract

A yellow fluorescent protein (YFP) present in a strain of bioluminescent bacteria is shown here not only to modify the color and intensity of the emission, as already known and attributed to the interaction of YFP with a luciferase intermediate, but also 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 degrees C, at approximately 1 microM YFP concentration. Several hypotheses are considered to explain this effect, based either on inactivation of YFP itself at higher temperatures 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 temperatures, 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 temperatures, within a physiological range.

Time‐Resolved Fluorescence Study of the Dissociation of FMN from the Yellow Fluorescence Protein from Vibrio fischeri

“…Our binding results are fuHy in line with the data and conclusions of Sirokman et al (10), which were derived from steady-state fluorescence anisotropy measurements.…”

“…The equilibrium constant for the dissociation of FMN from YFP can be obtained from fluorescence parameters. According to Sirokman et al (10) and Spencer and Weber (15), the following equations are used for determination of K o from steady-state anisotropies <r>:…”

“…The YFP has these convenient properties as weH (10,11). It is one of the flavoproteins with the highest fluorescence quantum yield occurring in nature and it is characterized by a peculiarly red-shifted fluorescence spectrum as compared to that of FMN in aqueous solution.…”

Two Lysine Residues in the Bacterial Luciferase Mobile Loop Stabilize Reaction Intermediates

“…We observed a curved plot indicative of a binding event. Iodide is also believed to quench the intermediate(s) that generate the excited state emitter (36). The decay rate of bioluminescence was significantly increased in the presence of iodide for K283A but only slightly for K286A (Fig.…”

“…Collisional Quenching-The experiments were conducted at 20°C essentially as described (36). Decay rates and initial velocities were determined from samples containing ϳ1 M luciferase and an appropriate concentration of KCl to maintain a constant ionic strength of 250 mM.…”