Signalling

Part:BBa_K212006

Designed by: Elias Scheer   Group: iGEM09_Brown   (2009-10-20)

mRBP (modified ribose binding protein - now binds histamine)

This is a mutated version of RBP, an E. coli periplasmic ribose-binding protein. Natively, ribose binds as a ligand, transducing a message to the trg domain of a transmembrane histidine kinase. Here we have computationally redesigned RBP's ligand binding pocket to bind histamine preferentially over ribose, yielding mRBP.

Usage and Biology

There are sixteen locations (S9K, N13D, F15E, F16H, N64H, D89G, R90D, S103T, I132D, A137T, R141L, F164D, N190H, F214V, D215G, Q235T) at which RBP is mutated to create mRBP. These mutations redesigned the protein's ligand-binding pocket to bind to histamine instead of ribose, while still allowing the protein to fold properly and maintain its shape. These mutations were introduced based on predictions made by the Rosetta 3 macromolecular modeling program, which probabilistically searches through mutations to RBP's ligand binding pocket, then simulating the interaction between the mutated protein and histamine (similar to the method in Looger et al.). The predicted design with the lowest energy was selected and the DNA that coded for it was synthesized by GeneArt (see Brown 2009 wiki>Projects>Histamine Binding for more details).

The mechanism of mRBP's binding may be thought of like that of a venus fly-trap. There are two domains that form the top and bottom of its "mouth", and the ligand binding pocket is between these two domains. The two domains are connected by a hinge that allows the protein to snap shut on a ligand when it goes into its bound conformation.

MRBP schematic.png

Adapted from Looger et al Fig 3 In its bound conformation, mRBP interacts with the Trg domain of the chimeric TrgEnvZ histidine kinase, which then phosphorylates OmpR, which causes expression of genes under the OmpC promoter. The more mRBPs exist in the periplasmic domain bound to histamine, the more it interacts with TrgEnvZ, and the more gene expression there is in a cell containing all three genes and the promoter.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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