Structure and function of the ribose-binding protein of Escherichia coli; surfaces which mediate transport and chemotaxis

Robert Alan Binnie, Purdue University

Abstract

The rbs operon (rbsDACBKR) of Escherichia coli encodes the ribose high-affinity membrane transport system, a repressor which regulates transcription of the operon, ribokinase, the cytoplasmic enzyme which catalyzes the first step in metabolism of D-ribose, and a protein of unknown function. The high-affinity transport system is comprised of two inner membrane-associated proteins and the soluble, periplasmic ribose-binding protein (RBP). In addition to its role in membrane transport, RBP also serves as the primary receptor for chemotaxis towards ribose. Liganded binding protein interacts with the chemotactic transducer Trg to communicate changes in the concentration of extracellular ribose to the interior of the cell. In order to predict which parts of RBP might be involved in the chemotactic interaction, an algorithm based on the similarities and differences of RBP and two related binding proteins was used. By selecting segments of RBP amino acid sequence which are similar to those found in galactose-binding protein (which shares the Trg interaction with RBP) but dissimilar to those found in arabinose-binding protein (which folds like RBP and galactose-binding protein, but does not interact with Trg) six stretches of sequence proposed to be likely to interact with Trg were found. The gene for the binding protein was deleted from the chromosome and high affinity transport and chemotaxis was reconstituted by the introduction of a phagemid encoding RBP. Using this system, a large collection of mutations were created at solvent-exposed sites on the exterior of RBP. In vivo and in vitro characterization of these mutant RBP's has provided a functional map of the surface RBP. Analysis of the phenotypes of these mutant proteins shows that the area of ribose-binding protein which interacts with the transport complex is located on the face of the protein opposite the hinge. Mutations affecting chemotaxis are found on two separate areas on the surface of the molecule. One of these areas is composed of a subset of the zone involved in transport. The other area implicated for chemotactic function is adjacent to the hinge on the face of the molecule opposite from the surface involved in transport.

Degree

Ph.D.

Advisors

Hermodson, Purdue University.

Subject Area

Biochemistry|Molecular biology

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