Photocontrolled Reversible Binding between the Protein A-Derived Z Domain and Immunoglobulin G

Photoisomerization of the trans and cis isomers of azobenzene derivatives has been used to control the function of biomolecules in a reversible and nondestructive manner. In this study, affibody molecules, representing a class of small, helical proteins that can be engineered for binding to a wide range of target proteins, have been investigated by the incorporation of a photoswitchable azobenzene derivative in the molecule. Three different Z domain variants were produced by solid phase peptide synthesis and conjugated by thiol-directed chemistry to an azobenzene-based photoswitch. The proteins were screened for binding to and light elution from an IgG-sepharose affinity column. One of the tested Z variants, Z_C3, showed efficient binding to the column and could be eluted by irradiation with light at 400 nm. In a reverse affinity chromatography assay, where the Z_C3 variant was coupled to sepharose, human IgG1 could be captured to the column and partially eluted by light. Further studies of the azobenzene-conjugated Z_C3 domain by surface plasmon resonance (SPR) confirmed the high affinity binding to IgG, and circular dichroism (CD) spectroscopy showed that the protein has a high α-helical secondary structure content.