Out with the Old, In with the New

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Science  18 May 2001:
Vol. 292, Issue 5520, pp. 1263
DOI: 10.1126/science.292.5520.1263c

Maltose binding protein (MBP), a periplasmic binding protein from Escherichia coli, has a ligand binding site located at the interface of two domains. Maltose binding initiates a conformational change from an open to a closed state, and earlier work has demonstrated coupling of this change to a fluorescent readout.

Marvin and Hellinga have used rational design to convert MBP into a zinc sensor. Computational analysis predicted twenty potential tetrahedral coordination sites within the interdomain interface. Four of these modified MBPs were engineered: two by altering maltose binding residues (A sites) and two that left the maltose binding site intact (B sites). All four proteins bound zinc, but only the A sites coupled zinc binding to formation of the closed state. A hybrid with both an A and a B site exhibited increased zinc affinity. The affinity also could be improved by refining the coordination sphere or by making mutations designed to favor the closed state. Finally, a substantial enhancement in affinity was achieved by eliminating polar residues that would have formed hydrogen bonds with maltose and that would be vestigial in the zinc sensor. — VV

Proc. Natl. Acad. Sci. U.S.A.98, 4955 (2001).

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