| The introduction of new functions into proteins and the redesign of ligand-binding specificity has been the focus of many previous works. However, the design of two intercommunicating sites has not been heavily pursued, and is the primary focus of this work. Analysis of the structural principles of conformational coupling suggests that it is possible to establish allosteric linkage between two sites in any protein in which at least two rigid units move relative to each other. The most straightforward manner by which this can be achieved is through a direct coupling mechanism, wherein both sites are located at the interface of two such subunits and are comprised of residues contributed by each. This theory also predicts that it is possible to control the equilibrium activity of one function by manipulating global conformational equilibria. This can be achieved through modification of an allosterically linked site, analogous to the control exerted by allosteric effectors in natural systems. Furthermore, spatial separation of allosterically linked sites should allow the microscopic activities of each to be independently manipulated.;These predictions have been experimentally validated by demonstration that: (1) the emission intensity of fluorophores, covalently attached to predicted "proto-allosteric" sites in both maltose-binding protein (MEBP) and glucose-binding protein is heterotropically cooperative with respect to ligand binding; (2) observed ligand-binding affinities in MBP may be tuned by manipulating conformational equilibria through mutation of allosterically coupled sites; and (3) it is possible to radically change the specificity of MBP by converting it into a zinc sensor through structure-based computational design. |
