Structural Basis of the Light-Switchable Interaction between an Azobenzene Side Chain in a Biosynthetic Protein and...

Area of Science:

• Protein science
• Biochemistry
• Structural biology

Background:

• Azobenzene derivatives enable light-induced reversible trans↔cis isomerization, gaining attention in protein science.
• p-(Phenylazo)-L-phenylalanine (Pap) is used in the Azo-tag system for light-controlled protein purification via supramolecular complex formation with α-cyclodextrin (α-CD).
• The Pap side chain in its trans-configuration binds to α-CD, while cis-isomerization causes dissociation.

Purpose of the Study:

• To elucidate the X-ray crystal structure of super-folder green fluorescent protein (sfGFP) with surface-displayed Pap in complex with α-CD.
• To understand the molecular interactions and binding modes between Pap and α-CD within a protein context.
• To provide insights for future protein engineering of light-controllable functions.

Main Methods:

• X-ray crystallography was used to analyze the complex of sfGFP displaying Pap at position 39 with α-CD.
• Structural analysis focused on the host-guest interaction between Pap and α-CD.
• Comparison of experimental structural data with molecular modeling predictions.

Main Results:

• The crystal structure confirmed the general mode of host-guest interaction between Pap and α-CD.
• Unexpectedly, α-CD bound via its narrow end to the aminoacyl moiety of Pap.
• The azobenzene side chain of Pap exhibited a twisted conformation, deviating from the expected planar arrangement.

Conclusions:

• The determined crystal structure offers valuable insights into the binding of photoswitchable noncanonical amino acids within proteins.
• The findings reveal specific binding orientations and conformational adaptations of Pap and α-CD.
• This structural information is crucial for advancing the molecular engineering of proteins with light-controllable functionalities.