Detection of protein-protein interactions in the alkanesulfonate monooxygenase system from Escherichia coli

Abstract

The two-component alkanesulfonate monooxygenase system utilizes reduced flavin as a substrate to catalyze a unique desulfonation reaction during times of sulfur starvation. The importance of protein-protein interactions in the mechanism of flavin transfer was analyzed in these studies. The results from affinity chromatography and cross-linking experiments support the formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD). Interactions between the two proteins do not lead to overall conformational changes in protein structure, as indicated by the results from circular dichroism spectroscopy in the far-UV region. However, subtle changes in the flavin environment of FMN-bound SsuE that occur in the presence of SsuD were identified by circular dichroism spectroscopy in the visible region. These data are supported by the results from fluorescent spectroscopy experiments, where a dissociation constant of 0.0022 +/- 0.0010 muM was obtained for the binding of SsuE to SsuD. Based on these studies, the stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD.

FIG. 1.

Mechanism of desulfonation by the two-component alkanesulfonate monooxygenase system from E. coli.

FIG. 2.

SDS-PAGE (10% acrylamide) from affinity chromatography experiments with SsuE and His-tagged SsuD. Lane 1, protein standard marker; lane 2, native SsuD; lane 3, native SsuE; lane 4, protein fractions collected after washing with 300 mM imidazole buffer containing SsuE and His-tagged SsuD.

FIG. 3.

Far-UV CD spectroscopy of SsuE and SsuD. The spectrum of SsuE (solid line) was obtained with 2.4 μM SsuE in 25 mM phosphate buffer (pH 7.5) at 25°C. The SsuD spectrum (dotted line) was obtained with 1.2 μM SsuD in 25 mM phosphate buffer (pH 7.5) at 25°C. The SsuE/SsuD spectrum (dashed line) was obtained with 2.4 μM of SsuE and 1.2 μM SsuD. The additive spectrum was from individual spectra of SsuE and SsuD (dashed line). Measurements were taken in 0.1-nm increments from 300 to 185 nm in a 0.1-cm-path-length cuvette. Each spectrum is the average of four scans; smoothing of the data was performed using the default parameters within the Jasco J-720 software.

FIG. 4.

Visible CD spectra for FMN in the absence and presence of SsuE and SsuD. The spectrum of FMN (solid line) was obtained with 20 μM FMN in 25 mM potassium phosphate buffer (2.0-ml total volume) (pH 7.5), 10% glycerol, and 100 mM NaCl at 25°C. The FMN/SsuE spectrum (dashed line) was obtained with 40 nmol of FMN and 20 μM of SsuE in 25 mM potassium phosphate buffer (2.0-ml total volume) (pH 7.5), 10% glycerol, and 100 mM NaCl at 25°C. The FMN/SsuE/SsuD spectrum (dotted line) was obtained with a 1:1 stoichiometric addition of 20 μM SsuD to 40 μM FMN-bound SsuE (2.0-ml total volume). Measurements were taken in 0.2-nm increments from 550 to 300 nm in a 1-cm-path-length cuvette. Each spectrum is the average of eight scans; smoothing of the data was performed using the default parameters within the Jasco J-720 software.

FIG. 5.

Chemical cross-linking of SsuE and SsuD. (A) structure of the trifunctional cross-linker reagent (ProFound Label Transfer Sulfo-SBED). The reagent contains an amine reactive site, a photoreactive arylazide, a biotin label, and a cleavable disulfide bond. (B) Silver-stained SDS-PAGE (10% acrylamide) from cross-linking reactions. Lanes 1 and 6, protein markers; lane 2, SsuE and SsuD proteins; lane 3, treated SsuE; lane 4, cross-linked SsuE and SsuD; lane 5, DTT-treated cross-linked SsuE and SsuD; lane 7, native SsuE and SsuD. The samples were mixed with nondenaturing and nonreducing sample buffer except for protein markers (lanes 1 and 6) and SsuE and SsuD proteins (lane 2).

FIG. 6.

Fluorometric titration of SsuD to FMN-bound SsuE. (A) Emission intensity of fluorometric titration of FMN-bound SsuE with SsuD. A 0.40 μM concentration of FMN-bound SsuE was titrated with between 0.02 and 0.95 μM SsuD. Emission intensity measurements from 470 to 650 nm were made using excitation at 450 nm. (B) The concentration of SsuD bound was plotted against the total SsuD and was fit to equation 1 (see text) in experimental procedures using the Kaleidagraph software (Abelbeck Software, Reading, PA).

FIG. 7.

UV-visible spectra for filtration of FMN in the presence of SsuE and SsuD proteins. The flavin spectra were obtained with 20 μM FMN in 25 mM phosphate buffer (pH 7.5), 10% glycerol, and 100 mM NaCl at 25°C in the presence of 20 μM of SsuE and SsuD proteins in a 2.0-ml total volume. The spectra were taken before filtration (solid line) and after filtration (dashed line) on a UV-visible spectrophotometer at a wavelength of 450 nm.