Use of Mass Spectral Disassembly to Elucidate the Quaternary Structure of the Complex Heterotetrameric Sarcosine Oxidase
EMSL Project ID
2176
Abstract
The measurement of intact macromolecular assemblies and their disassembly in the mass spectrometer has recently emerged as a powerful new tool in determining protein quaternary structure. Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional protein that catalyzes both the oxidation of sarcosine (N-methylglycine) and the synthesis of 5,10-methylenetetrahydrofolate. TSOX contains four different subunits (alpha, beta, gamma, delta) that range in size from about 100 kDa to 10 kDa. The isolated enzyme contains three coenzymes (noncovalently bound FAD and NAD+ and covalently bound FMN) and a binding site for a fourth coenzyme (tetrahydrofolate) that acts as a substrate. The 8alpha-position of the FMN ring is attached to His173 in the beta subunit of TSOX. In collaboration with Ljiljana Tolic and Richard Smith, we recently established conditions which permit ionization of TSOX in an apparently native-like conformation. In these studies, intact TSOX with all of its associated coenzymes was detected using electrospray ionization with a 7-tesla FTICR spectrometer. The observed molecular mass (180,700 ? 300 u) was in good agreement with the theoretical value (180,602 u). The ability to measure the intact complex suggests that it may be possible to probe the structural organization of the multiple subunits and coenzymes by using gas phase collisions in the source to disassemble the complex. Results obtained in the proposed collision-induced dissociation studies will be compared with those predicted by a current model for TSOX that has been developed based on a variety of biochemical studies (see attachment).
Project Details
Project type
Exploratory Research
Start Date
2001-10-16
End Date
2003-06-18
Status
Closed
Released Data Link
Team
Principal Investigator