THE RATIONAL DESIGN OF ALLOSTERIC INTERACTIONS IN A MONOMERIC PROTEINAND ITS APPLICATIONS TO THE CONSTRUCTION OF BIOSENSORS

Citation
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
Citations number
32
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary Sciences
ISSN journal
0027-8424
Volume
94
Issue
9
Year of publication
1997
Pages
4366 - 4371
Database
ISI
SICI code
0027-8424(1997)94:9<4366:TRDOAI>2.0.ZU;2-V
Abstract
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.