@article {1561, title = {Conversion of a maltose receptor into a zinc biosensor by computational design.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {98}, year = {2001}, month = {2001 Apr 24}, pages = {4955-60}, abstract = {

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.

}, keywords = {Alanine, Allosteric Regulation, Bacterial Proteins, Binding Sites, Biosensing Techniques, Calorimetry, Carrier Proteins, Computer Simulation, Fluorescence, Kinetics, Ligands, Maltose, Maltose-Binding Proteins, Models, Molecular, Mutation, Protein Conformation, Protein Engineering, Signal Transduction, Software, Substrate Specificity, Temperature, Thermodynamics, Zinc}, issn = {0027-8424}, doi = {10.1073/pnas.091083898}, author = {Marvin, J S and Hellinga, H W} } @article {1560, title = {Manipulation of ligand binding affinity by exploitation of conformational coupling.}, journal = {Nature Structural Biology}, volume = {8}, year = {2001}, month = {2001 Sep 27}, pages = {795-8}, abstract = {

Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from an approximately 100-fold improvement (7.4 nM) to an approximately two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).

}, keywords = {Allosteric Regulation, Allosteric Site, Apoproteins, ATP-Binding Cassette Transporters, Calorimetry, Carrier Proteins, Escherichia coli, Escherichia coli Proteins, Kinetics, Ligands, Maltose, Maltose-Binding Proteins, Models, Molecular, Monosaccharide Transport Proteins, Mutation, Protein Binding, Protein Conformation, Protein Engineering, Temperature, Thermodynamics}, issn = {1072-8368}, doi = {10.1038/nsb0901-795}, author = {Marvin, J S and Hellinga, H W} } @article {1562, title = {Protein engineering and the development of generic biosensors.}, journal = {Trends in Biotechnology}, volume = {16}, year = {1998}, month = {1998 Apr}, pages = {183-9}, abstract = {

Biosensors exploit the remarkable specificity of biomolecular recognition to provide analytical tools that can measure the presence of a single molecular species in a complex mixture. A new strategy is emerging in the development of biosensor technologies: molecular-engineering techniques are being used to adapt the properties of proteins to simple, generic detector instrumentation, rather than adapting instruments to the unique requirements of a natural molecule.

}, keywords = {Binding Sites, Biosensing Techniques, Protein Binding, Protein Engineering, Proteins, Signal Transduction}, issn = {0167-7799}, author = {Hellinga, H W and Marvin, J S} } @article {1563, title = {The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {94}, year = {1997}, month = {1997 Apr 29}, pages = {4366-71}, 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 fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific 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 altered without affecting transduction of the binding event by fluorescence. 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.

}, keywords = {Allosteric Regulation, ATP-Binding Cassette Transporters, Binding Sites, Biosensing Techniques, Biotechnology, Carrier Proteins, Computer Simulation, Escherichia coli Proteins, Fluorescent Dyes, Maltose, Maltose-Binding Proteins, Models, Molecular, Monosaccharide Transport Proteins, Mutation, Protein Conformation}, issn = {0027-8424}, author = {Marvin, J S and Corcoran, E E and Hattangadi, N A and Zhang, J V and Gere, S A and Hellinga, H W} }