Genetically encoding photoswitchable click amino acids for general optical control of conformation and function of proteins

Abstract

Over the past decade, photoswitchable molecules have been emerging as attractive tools for investigating biological processes with spatiotemporal resolution in a minimally invasive fashion. Photoswitches built on light-sensitive proteins or domains have significantly advanced neuronal and cellular studies. To install photosensitivity to general proteins and to enable high specificity for modulation, photoswitchable click amino acids (PSCaas) based on azobenzene have been developed and recently genetically incorporated into proteins via the expansion of the genetic code. PSCaas allow targeting selected sites in a protein for high specificity and are generally applicable to various proteins. In addition, PSCaas contain a click functional group, which selectively reacts with an appropriately positioned cysteine forming a photocontrollable bridge on the protein in situ. The photocontrollable bridge enables reversible modulation of the secondary structure of the spanned region and thus the function of the protein. In this chapter we describe the design and genetic encoding of PSCaa. Protocols are presented for incorporating PSCaa into a model protein calmodulin to build the bridge followed by photocontrol of calmodulin's conformation and binding function.

Visible-Light-Mediated Modification and Manipulation of Biomacromolecules

“…As demonstrated in the previous examples, precise and minimally invasive control over protein structure and activity may be achieved with a photoswitchable amino acid at a single site . However, the relatively small conformational changes induced by photoswitchable amino acids may be insufficient to recapitulate larger-scale structural changes that are crucial to many biological processes. , To address this shortcoming, a well-established alternative approach is to link two suitably positioned cysteine residues in the protein or peptide together with a linker that contains an azobenzene moiety, which generates a photoswitchable bridge . Photoisomerization of the azobenzene bridge alters its length, which may significantly perturb the protein’s secondary structure.…”

“…To circumvent these issues, a cysteine cross-link can be established using a genetically encoded photoswitchable click amino acid (PSCaa, 359 ) (Scheme ). Pioneered by Hoppmann, Wang, and co-workers, ,− PSCaa 359 are derivatives of AzoPhe ( 356 ) containing a reactive handlesuch as an alkene, benzyl chloride, or keto groupthat enables the site-specific formation of a photoswitchable bridge. In 2015, Wang and co-workers incorporated a pentafluoroazobenzene-based click amino acid (F–PSCaa, 360 ) into calmodulin (CaM), a calcium-binding messenger protein, in E.…”

Synthetic Biology Meets Ca2+ Release-Activated Ca2+ Channel-Dependent Immunomodulation

“…While several photosensory tools can be controlled in a reversible manner, chemical dimerizers, proteolytic cleavage, or photocrosslinking UAAs can only be operated irreversibly. Among photosensory UAAs, photoswitchable ones have been synthesized and shown to be suitable for reversible manipulation of protein function using two alternating wavelengths [ 305 , 306 , 375 ]. This, however, has so far only been accomplished for calmodulin [ 306 , 375 ] and glutamate receptors [ 307 ].…”

Applications of genetic code expansion technology in eukaryotes

“…To overcome these limitations, photoswitchable amino acids (PSAAs) have been developed. PSAAs containing an azobenzene moiety can covalently bind to a cysteine residue in the protein domain to form a photocontrollable bridge in situ , allowing light-inducible control of protein conformation ( Hoppmann et al, 2015 ; Hoppmann and Wang, 2019 ). Furthermore, the azobenzene moiety in a PSAA can reversibly change its conformation under two wavelengths of light, making the regulation of protein activity reversible ( Aemissegger and Hilvert, 2007 ).…”