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 reversi bility of biomolecular interactions. We have previously demonstrated that s timuli-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 allo wed a mechanistic investigation of the a mart polymer switches. This polyme r was conjugated via a vinyl sulfone terminus to cysteine residues of genet ically engineered streptavidin mutant E116C, where the polymer is conjugate d close to the biotin-binding site, and streptavidin mutant S139C, where th e conjugation site is distant. The biotin binding switching activity was st rongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small eff ects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biot in with the E116C conjugate was due to the blocking the reassociation of bi otin. The addition of free polymer to purified E116C conjugates was also sh own to increase the blocking and release properties of the switch. This eff ect was site dependent, suggesting that the conjugated polymers were direct ing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight th at can be utilized to design better molecular switches for a variety of sti muli-responsive polymer-protein conjugates.