A guide to designing photocontrol in proteins: methods, strategies and applications

Abstract

Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase

“…IC 50 values determined in the indirect assays indicated that catalytic activity could be regulated about 15-fold by this approach, whereas the direct assay indicated a light-regulation factor of about 5. These values compare favorably with most light-regulation factors reported in the literature and underline the versatility and efficiency of “photoxenoprotein” approaches …”

“…These values compare favorably with most light-regulation factors reported in the literature and underline the versatility and efficiency of "photoxenoprotein" approaches. 1 ■ METHODS Strains, Cloning, and Site-Directed Mutagenesis. E. coli BL21 Gold (DE3) and E. coli NEB Turbo cells were used as hosts for protein expression and molecular cloning, respectively.…”

“…Hence, light-driven biocatalysis has become an emerging topic in the fields of synthetic biology, biotherapeutics, and biotechnology. The used techniques can be divided into photopharmacology, which is based on small-molecule photosensitive effectors of enzyme function, hybrid protein optogenetics, which describes the fusion of an enzyme with a photoreactive biological module, and photoxenoprotein engineering, which means the site-specific incorporation of a photoresponsive noncanonical amino acid (ncAA) into an enzyme . Traditionally, ncAAs have been introduced into proteins through chemical modification of individual amino acids with reactive side chains .…”

Peptide Conjugated Dihydroazulene/Vinylheptafulvene Photoswitches in Aqueous Environment

“…Specifically, the light‐induced change in electronic properties and geometry of the photoresponsive core structure can be harvested to regulate the properties of a bioactive molecule of choice. For instance, photoswitches were applied as light‐controllable antibiotics, [8,9] DNA‐binders, [10,11] microtubule modulators, [12,13] and as enzyme inhibitors [14–16] . Moreover, the large structural change in geometry of the photoswitchable unit can be translated onto a larger structure, such as a biomacromolecule, when incorporated in a strategically suitable position [1] .…”

“…[14][15][16] Moreover, the large structural change in geome-try of the photoswitchable unit can be translated onto a larger structure, such as a biomacromolecule, when incorporated in a strategically suitable position. [1] Thus, it can be used to efficiently alter the properties of, for instance, nucleic acids, [17] peptides, [18] and proteins [14] by use of light as external stimulus. Photoswitches such as (heteroaryl) azobenzenes, [19,20] diarylethenes, [21] and spiropyrans [22] have been studied previously in biological contexts.…”