A noncovalent switch for lysozyme.

A new concept for the external control of protein activity is presented and demonstrated on the example of an artificial Lysozyme switch. Radical copolymerization of selected methacrylamide-based comonomer units tailored for amino acid residues surrounding the active site furnishes polymeric protein hosts that are able to inhibit enzymatic activity in a highly efficient dose-dependent manner (IC50 approximately 1.0 equiv approximately 0.7 microM). All binding site types on the polymer work cooperatively, using electrostatic attraction, hydrophobic forces, and substrate mimicry. In a native gel electrophoresis, the well-defined 2:1 complex (polymer/protein) migrates to the anode. Even at 250 mM NaCl, a 10-fold polymer excess is able to shut down bacterial cell wall degradation completely. A kinetic investigation points to a competitive mechanism (Lineweaver-Burk plots). CD spectra of pure Lysozyme and its polymer complex are indistinguishable; together with a total lack of preincubation time for maximum inhibition, experimental evidence is thus produced for a preserved tertiary enzyme structure-no denaturation occurs. Addition of the superior complexing agent polyarginine to the enzyme-polymer complex mildly detaches the inhibitor from the protein surface and leads to 90% recovery of enzymatic activity. Thus, Lysozyme could be turned off, on, and off again by consecutive addition of the polymeric inhibitor, polyarginine, and polymer again.