Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield

Many medical and biotechnological processes rely on controlling and manipul ating the molecular-recognition capabilities of proteins(1-4). This can be achieved using small molecules capable of competing for protein binding or by changing environmental parameters that affect protein structure and henc e binding. An alternative is provided by stimuli-responsive polymers that c hange reversibly from a water-soluble expanded coil to a water-insoluble co llapsed globule upon small changes in temperature, pH or light intensity: w hen attached to proteins in the vicinity of their binding sites, they rever sibly block and release small ligands(1,5-7). Here we show how this approac h can be extended to achieve size-selective binding of large, macromolecula r ligands. We use the thermally responsive polymer poly(N,N-diethylacrylami de) (PDEAAm), and attach it to the protein streptavidin approximately 20 An gstrom from the binding site for biotinylated proteins. Below the lower cri tical solution temperature of PDEAAm, the polymer is in its extended state and acts as a 'shield' to block the binding of large biotinylated proteins; above this temperature, it collapses and exposes the binding site, thereby allowing binding. We rnd that the degree of shielding depends on both the size of the biotinylated protein and the size of PDEAAm, suggesting that 's mart' polymer shields could be tailored to achieve a wide range of size-dep endent ligand discrimination for use in affinity separations, biosensors an d diagnostics technologies.