Mechanistic investigation of smart polymer-protein conjugates.

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.