Abstact

Azobenzene derivatives, which show light-induced reversible trans<-->cis isomerization, have gained increasing attention in the area of protein science. p-(Phenylazo)-L-phenylalanine (Pap) was recently employed to enable the light-controlled affinity purification of biosynthetic proteins as part of the Azo-tag. Specific supramolecular complex formation with immobilized alpha-cyclodextrin (alpha-CD) groups is mediated by the Pap side chain in its low-energy trans-configuration, whereas photoisomerization to the cis-state leads to immediate dissociation. Here, we describe the X-ray crystallographic analysis of super-folder green fluorescent protein (sfGFP) displaying Pap at amino acid position 39 on its surface in complex with alpha-CD. While this experimental structure generally confirms the mode of host-guest interaction predicted by molecular modeling, there are two unexpected observations: (i) the conically shaped alpha-CD binds with its narrow end toward the aminoacyl moiety of Pap, despite appearing sterically more demanding, and (ii) the azobenzene side chain shows a considerably twisted conformation of its two phenyl rings, which contrasts with the fully coplanar arrangement usually anticipated for unmodified azobenzene and its chemical derivatives. Thus, this crystal structure of the photoswitchable noncanonical amino acid Pap (also known as AzoF or AzoPhe) provides valuable insight for future molecular engineering endeavors to endow proteins with light-controllable functions.