Rational design, production and in vitro analysis of photoxenoproteins

Hiefinger, Caroline; Mandl, Sabrina; Wieland, Mona; Kneuttinger, Andrea Christa

doi:10.1016/bs.mie.2022.12.003

Cited by 2 publications

(4 citation statements)

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“…As there are few examples of ribosomal flexibility, recently two ncAAs with large side chains, o -nitrobenzyl- O -tyrosine, and phenylalanine-4′-azobenzene have been introduced for light-sensitive manipulation of proteins, termed by their designers “photoxenoproteins” (Figure : 4 , 5 ) . It is difficult to predict precisely what groups will fit in the active site of the ribosome or through the exit tunnel, as some R groups less reminiscent of canonical amino acids are not incorporated, such as a tert -butyl side chain, while even bulkier ring systems, such as coumaryl, benzoyl, dansyl, and pyrenyl modifications to natural R groups as well as O -glycosylated side chains, have been incorporated despite hypothesized steric hindrance (Figure : 1 , 2 , 3 , 7 , 9 ). ,− In general, there have been few successful incorporations of side chains containing more than two coupled aromatic rings. , That said, Sisido and Hohsaka have integrated even larger structures into proteins, but N -modified diaminopropionic acids with quadruple and quintuple aromatic ring fluorescent group attachments were unsuccessful .…”

Section: Wild-type Ribosomes and The Insertion Of Noncanonical Amino ...mentioning

confidence: 99%

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Ishida,

Ngo,

Gundlach

et al. 2024

Chem. Rev.

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The introduction of noncanonical amino acids into proteins has enabled researchers to modify fundamental physicochemical and functional properties of proteins. While the alteration of the genetic code, via the introduction of orthogonal aminoacyl-tRNA synthetase:tRNA pairs, has driven many of these efforts, the various components involved in the process of translation are important for the development of new genetic codes. In this review, we will focus on recent advances in engineering ribosomal machinery for noncanonical amino acid incorporation and genetic code modification. The engineering of the ribosome itself will be considered, as well as the many factors that interact closely with the ribosome, including both tRNAs and accessory factors, such as the all-important EF-Tu. Given the success of genome re-engineering efforts, future paths for radical alterations of the genetic code will require more expansive alterations in the translation machinery.

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Section: Wild-type Ribosomes and The Insertion Of Noncanonical Amino ...mentioning

confidence: 99%

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Ishida,

Ngo,

Gundlach

et al. 2024

Chem. Rev.

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“…incorporation site is marked by an amber codon in the genetic code of the enzyme and AzoF is co-translationally incorporated by using a heterologous, orthogonal aminoacyl-tRNA-synthetase/tRNA (aaRS/tRNA) pair. 26,27 AzoF is often used to engineer light-sensitive proteins, known as photoxenoproteins. 26,28 Recently, we have achieved to control enzyme activity by a factor of ~10-fold in the allosteric bi-enzyme complex imidazole glycerol phosphate synthase (ImGPS) using the configurational switch of AzoF.…”

Section: Equationmentioning

confidence: 99%

“…26,27 AzoF is often used to engineer light-sensitive proteins, known as photoxenoproteins. 26,28 Recently, we have achieved to control enzyme activity by a factor of ~10-fold in the allosteric bi-enzyme complex imidazole glycerol phosphate synthase (ImGPS) using the configurational switch of AzoF. 29,30 ImGPS belongs to the family of glutamine amidotransferases consisting of a glutaminase subunit, HisH, and a cyclase subunit, HisF (SI part1: Figure S1).…”

Section: Equationmentioning

confidence: 99%

“…Figure 6 and Figure 7). 26 For this, we initiated the reaction by the addition of ImGPS, followed the decrease of PRFAR turnover in the dark for ~5 min and then started the irradiation while following PRFAR turnover until the reaction stopped. This allowed us to detect effects that are primarily caused by the injection of VE at 𝑃𝑆𝑆 , 𝑃𝑆𝑆 or 𝑃𝑆𝑆1 / , as these states were reached after a maximum of 20 s (cf.…”

Section: Comparison Of Moderate and Low Vet Effects With E↔z Switchin...mentioning

confidence: 99%

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Promoting enzyme catalysis via azobenzene facilitated vibrational energy transfer

Lahmy,

Hiefinger,

Zeqiri

et al. 2024

Preprint

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Besides its capability to switch between two configurations, azobenzene converts light energy to vibrational energy (VE). Incorporation of the azobenzene-based unnatural amino acid AzoF can, hence, facilitate the transfer of VE into an enzyme. We were interested whether this VE transfer might increase conformational dynamics important for catalysis to ultimately promote enzyme activity. To this end, allosteric enzymes are well-suited model systems since allosteric regulation is mediated by conformational dynamics. To promote catalysis via VE transfer, we selected two positions for incorporation of AzoF in the prominent allosteric bi-enzyme complex imidazole glycerol phosphate synthase (ImGPS). While W123 in the HisH subunit is close to the critical active site and a key player for allostery, S55 in the HisF subunit is more distant contributing only moderately to allostery, as shown via in-depth biophysical and computational evaluations. We further demon-strated that simultaneous irradiation of AzoF with 365 nm and 420 nm produces higher amounts of VE than monochromatic irradiation and results in the appearance of a third spectroscopic signal originating from the azobenzene moiety. Remarkably, enzyme activity increased upon dichromatic irradiation for ImGPS(W123AzoF) but not for wild-type ImGPS or ImGPS(S55AzoF) and returned to initial activity values in the dark indicating that the change in activity was caused by VE transfer. Our findings provide a proof-of-concept for promoting enzyme activity by VE transfer and open up further possibilities to study the relationship between dynamics and enzyme catalysis.

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Rational design, production and in vitro analysis of photoxenoproteins

Cited by 2 publications

References 86 publications

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Engineering Ribosomal Machinery for Noncanonical Amino Acid Incorporation

Promoting enzyme catalysis via azobenzene facilitated vibrational energy transfer

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