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

@article{Marvin1997, 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.}, address = {Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.}, author = {Marvin, J. S. and Corcoran, E. E. and Hattangadi, N. A. and Zhang, J. V. and Gere, S. A. and Hellinga, H. W. }, citeulike-article-id = {2717133}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {biosensor, computational\_protein\_design}, month = {April}, number = {9}, pages = {4366--4371}, posted-at = {2008-04-25 10:19:53}, priority = {2}, title = {The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9113995}, volume = {94}, year = {1997} }

TY - JOUR ID - Marvin1997 TI - The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors. JF - Proceedings of the National Academy of Sciences of the United States of America VL - 94 IS - 9 SP - 4366 EP - 4371 AD - Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA. SN - 0027-8424 N2 - 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. KW - biosensor KW - computational_protein_design AU - Marvin, JS AU - Corcoran, EE AU - Hattangadi, NA AU - Zhang, JV AU - Gere, SA AU - Hellinga, HW PY - 1997/04/29/ UR - http://view.ncbi.nlm.nih.gov/pubmed/9113995 ER -