The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors

Abstract

Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system. We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site. Environmentally sensitive f luorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions. The f luorescence 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 altered without affecting transduction of the binding event by f luorescence. To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.Three phenomena illustrate the remarkable degree of functional control displayed by some proteins. First, structurally dissimilar (''allosteric'') ligands can influence the activity of one another. Such allosteric interactions are responsible for controlling most metabolic and cellular signal transduction pathways and therefore play a central role in regulating cellular physiology (1). Second, some proteins bind their ligands sigmoidally, which results in a transition between fully bound and ligand-free forms over a relatively short concentration range. This allows exquisite control over ligand loading, as illustrated by the efficient transport by hemoglobin of oxygen between tissues with high partial oxygen pressure to metabolically active, oxygen-starved tissues. Finally, some proteins are capable of exerting action at a distance. For instance, the binding of a hormone to a receptor at one side of a membrane results in a change of receptor activity at the other side. One of the great triumphs of molecular biology has been the unification of these three apparently disparate phenomena into a single theory of cooperative interactions between binding sites (2-4). Here we demonstrate how a rational design strategy can be derived from simple structural principles to introduce a heterotropically cooperative interaction between ligand binding at one site and activity at another site (in this case, the fluorescence of a fluorophore) in a monomeric protein, Escherichia c...

Conversion of a maltose receptor into a zinc biosensor by computational design

“…The cloning vector used for expressing MBP is a variant of the described vector (32), lacking a C-terminal oligohistidine peptide. Oligonucleotide-directed mutagenesis was carried out as described (32). Proteins were produced by induction with isopropyl ␤-Dthiogalactoside (IPTG) as described (32).…”

“…In this way allosteric control and cooperative ligand binding have evolved in some of the members of this protein superfamily (29). Conformational coupling has also been exploited to design biosensors (1) in which ligand binding is linked to an optical or electrochemical response in several bacterial periplasmic binding proteins (30,31), including MBP (32,33).…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Importantly, mutations in this region are distant from the site of substrate binding and are not likely to perturb binding directly. Placement of a fluorophore at position 95 in this interface results in a strong fluorescence increase upon maltose binding and closure (304), which was later traced to the loss of specific hydrogen-bonding interactions between the fluorophore and two tyrosine residues upon opening of the balancing interface (90). Insertion of residues with side chains of increasing size into the balancing interface created a set of maltose-BP mutants that bound maltose with increasing affinities (94,305), leading to the suggestion that the bulkier residues destabilized the open form and shifted the equilibrium from open to closed.…”

Extended Linear Response for Bioanalytical Applications Using Multiple Enzymes

“…In the particular example considered both criteria can be consistently satisfied. It is hoped that the developed ideas will stimulate additional research on bi-enzyme [23][24][25][26][27][28][29][30]39,[41][42][43] and other systems [44][45][46] with similar response characteristics.…”