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Transforming a bacterial protein into a zinc ‘biosensor’
By Bijal P. Trivedi

Scientists at Duke University Medical Center in Durham, North Carolina, have converted common proteins into sophisticated 'biosensors.' The technology could potentially be used to help detect and determine the levels of neurotransmitters in the brain, metal in drinking water, and explosives in ocean water.

Conversion of a maltose-binding protein (MBP) into a zinc sensor. View larger

Homme Hellinga, of the Department of Biochemistry, and a colleague altered the structure of a maltose-binding protein (MBP) from a bacterium so that it binds zinc instead of sugar. The project's ultimate goal is to modify MBP or MBP-like proteins to capture any desired target molecule. The binding of the protein to a target activates a fluorescent reporter molecule that is attached at another location on MBP. Measuring the amount of fluorescence indicates how many target molecules are present.

The region of the protein that actually binds the maltose is small, comprising about 20 amino acids. Using computational methods, Hellinga and Jonathan Marvin, also of the Department of Biochemistry, calculated the alterations in the position and type of the amino acids necessary to create a 'nook' with the right physical and chemical properties to attract a zinc atom.

The algorithms are not 'perfect,' but the computer can 'evolve' many more protein structures in silico than can be created in the laboratory. Hellinga says he can test more than a googol structures—a googol is the figure 1 followed by 100 zeros. Choosing several promising designs from billions and billions of structures, the researchers engineered the new proteins in the laboratory and tested their ability to bind zinc. One protein from this group undergoes another cycle of mathematical tweaking to improve its zinc-binding function.

Transforming MBP so that it binds zinc was half the challenge. The other half was retaining the mechanism in the protein for activating the fluorescent molecule. Hellinga incorporated a fluorescent molecule into MBP, so that the amount of fluorescent light increased when maltose was binding. MBP alone emits a soft blue glow, but when maltose binds the fluorescence changes to bright green-blue. Researchers can calculate precisely how much sugar is binding the protein by measuring the changes in fluorescence.

The Duke researchers have an ongoing project to develop a glucose sensor for use by diabetics. They are also developing proteins to detect the explosive TNT for use by the US Navy. The current work was supported by grants from the National Institutes of Health and the Office of Naval Research.

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Marvin, J.S. & Hellinga, H.W. Conversion of a maltose receptor into a zinc biosensor by computational design. Proc Natl Acad Sci USA 98, 4955-4960 (April 24, 2001).

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