Engineering the Maltose Binding Protein for Reagentless Fluorescence Sensing

Gilardi, Gianfranco; Zhou, L.Q.; Hibbert, Linda.; Cass, Anthony E. G.

doi:10.1021/ac00093a047

Cited by 144 publications

(96 citation statements)

References 14 publications

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“…This direct interaction has been used to measure ligand binding by a quasicompetitive, rather than allosteric, linkage method. Cysteine mutations have been constructed in the jaw region of both MBP (29) and its structural homologue, phosphate-binding protein (30). Environmentally sensitive fluorophores conjugated to these mutants respond to ligand but also increase the substrate K d substantially, indicating steric interference.…”

Section: Resultsmentioning

confidence: 99%

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

Marvin

Corcoran

Hattangadi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

180

139

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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...

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Section: Resultsmentioning

confidence: 99%

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

Marvin

Corcoran

Hattangadi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

180

139

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“…PBPs were successfully employed as binding moieties for biosensors by liganding with fluorophores (20,21). Although these biosensors are suitable for in vitro applications, liganded proteins are not suitable for in vivo imaging.…”

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confidence: 99%

Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors

Fehr¹,

Lalonde

Lager

et al. 2003

Journal of Biological Chemistry

279

257

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Glucose homeostasis is a function of glucose supply, transport across the plasma membrane, and metabolism. To monitor glucose dynamics in individual cells, a glucose nanosensor was developed by flanking the Escherichia coli periplasmic glucose/galactose-binding protein with two different green fluorescent protein variants. Upon binding of substrate the FLIPglu-170n sensor showed a concentration-dependent decrease in fluorescence resonance energy transfer between the attached chromophores with a binding affinity for glucose of 170 nM. Fluorescence resonance energy transfer measurements with different sugars indicated a broad selectivity for monosaccharides. An affinity mutant with a K d of ϳ600 M was generated, which showed higher substrate specificity, and thus allowed specific monitoring of reversible glucose dynamics in COS-7 cells in the physiological range. At external glucose concentrations between 0.5 and 10 mM, reflecting typical blood levels, free cytosolic glucose concentrations remained at ϳ50% of external levels. The removal of glucose lead to reduced glucose levels in the cell, demonstrating reversibility and visualizing homeostasis. Glucose levels dropped even in the presence of the transport inhibitor cytochalasin B, indicating rapid metabolism. Consistently, the addition of 2-deoxyglucose, which is not recognized by the sensor, affects glucose uptake and metabolism rates. Within the physiological range, glucose utilization, i.e. hexokinase activity, was not limiting. Furthermore, the results show that in COS-7 cells, cytosolic glucose concentrations can vary over at least two orders of magnitude. The glucose nanosensor provides a novel tool with numerous scientific, medical, and environmental applications.

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“…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).…”

mentioning

confidence: 99%

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

Marvin

Hellinga

2001

Proc. Natl. Acad. Sci. U.S.A.

150

114

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We have demonstrated that it is possible to radically change the specificity of maltose binding protein by converting it into a zinc sensor using a rational design approach. In this new molecular sensor, zinc binding is transduced into a readily detected fluorescence signal by use of an engineered conformational coupling mechanism linking ligand binding to reporter group response. An iterative progressive design strategy led to the construction of variants with increased zinc affinity by combining binding sites, optimizing the primary coordination sphere, and exploiting conformational equilibria. Intermediates in the design series show that the adaptive process involves both introduction and optimization of new functions and removal of adverse vestigial interactions. The latter demonstrates the importance of the rational design approach in uncovering cryptic phenomena in protein function, which cannot be revealed by the study of naturally evolved systems.

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Engineering the Maltose Binding Protein for Reagentless Fluorescence Sensing

Cited by 144 publications

References 14 publications

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

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

Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors

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

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