Biochemical and biophysical studies of the ribose transporter from Escherichia coli

Matthew C Clifton, Purdue University

Abstract

Sugar import mediated by ABC transporters is essential for the survival of prokaryotes such as Escherichia coli. Sugar import systems generally include a sugar binding protein, two transmembrane proteins, and two nucleotide binding proteins. In the ribose transport system, there are three proteins involved in the import mechanism. RbsB binds ribose in the periplasm and delivers it to the membrane protein a dimer of RbsC. The RbsC dimer binds to the cytoplasmic protein RbsA which encodes for two nucleotide binding domains. We have isolated three membrane protein complexes to investigate the effects of different cofactors in the transport cycle. The RbsAC complex bound to Mg-ADP includes a dimer of the transmembrane protein, RbsC, and the nucleotide binding protein RbsA. We have also isolated the vanadate trapped complex RbsABC Mg-ATP-VO4, which in addition to the RbsA and RbsC components, includes the ribose binding protein RbsB. Lastly we have isolated the RbsBC complex which includes a dimer of RbsC and the ribose binding protein RbsB. This represents the first time that any isolated ABC transporter complex has been reported in the absence of the nucleotide binding protein. Additional experiments were performed to understand how the presence or absence of individual proteins and cofactors control the stability of the complexes. Using the isolated complexes RbsAC, RbsABC, and RbsBC, as well as their individual components, we have identified an assembly and disassembly pathway for the ribose transport system that allows for the conversion of one complex to another in vitro. The individual proteins RbsA, RbsB, and RbsC as well as the full membrane protein complex, RbsABC, have been analyzed using analytical ultracentrifugation. To understand the stoichiometry of the membrane components, a new method has been developed to determine the amount of detergent bound to membrane proteins using both Rayleigh interference optics and absorption spectroscopy. This work demonstrates that RbsA and RbsB remain as monomers, the membrane protein RbsC forms a dimer, and the full ribose transport complex has the stoichiometry ABC2. Implications of the cofactor controlled complex formation and assembly and disassembly pathway are discussed.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biophysics|Biochemistry

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