Js. Marvin et al., THE RATIONAL DESIGN OF ALLOSTERIC INTERACTIONS IN A MONOMERIC PROTEINAND ITS APPLICATIONS TO THE CONSTRUCTION OF BIOSENSORS, Proceedings of the National Academy of Sciences of the United Statesof America, 94(9), 1997, pp. 4366-4371
Rational protein design is an emerging approach for testing general th
eories of structure and function, The ability to manipulate function r
ationally also offers the possibility of creating new proteins of biot
echnological value, Here we use the design approach to test the curren
t understanding of the structural principles of allosteric interaction
s in proteins and demonstrate how a simple allosteric system can form
the basis for the construction of a generic biosensor molecular engine
ering system, We have identified regions in Escherichia coli maltose-b
inding protein that are predicted to be allosterically linked to its m
altose-binding site, Environmentally sensitive fluorophores were coval
ently attached to unique thiols introduced by cysteine mutations at sp
ecific sites within these regions, The fluorescence of such conjugates
changes cooperatively with respect to maltose binding, as predicted,
Spatial separation of the binding site and reporter groups allows the
intrinsic properties of each to be manipulated independently, Provided
allosteric linkage is maintained, ligand binding can therefore be alt
ered without affecting transduction of the binding event by fluorescen
ce. To demonstrate applicability to biosensor technology, we hare intr
oduced a series of point mutations in the maltose-binding site that lo
wer the affinity of the protein for its ligand. These mutant proteins
have been combined in a composite biosensor capable of measuring subst
rate concentration within 5% accuracy over a concentration range spann
ing five orders of magnitude.