Side-chain specific isotopic labeling of proteins for infrared structural biology: The case of ring-D4-tyrosine isotope labeling of photoactive yellow protein

Rathod, Rachana; Kang, Zhouyang; Hartson, Steven D.; Kumauchi, Masato; Xie, Aihua; Hoff, Wouter D.

doi:10.1016/j.pep.2012.06.011

Cited by 8 publications

(9 citation statements)

References 49 publications

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“…To accomplish a quantitative incorporation of such slightly modified unnatural amino acid analogues, the endogenous tyrosine production has to be inhibited, because the natural tyrosine would compete successfully with the analogue. This may either be accomplished using selective inhibitors [95] or by genetic modification of the expression host [67]. As long as the corresponding tyrosyl tRNA-synthetase recognizes the analogue, a global substition of the natural amino acid by the analogue is achieved.…”

Section: Photoinduced Electron Transfer In Bluf Domains: Redox Tuningmentioning

confidence: 99%

Molecular eyes: proteins that transform light into biological information

2013

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Most biological photoreceptors are protein/cofactor complexes that induce a physiological reaction upon absorption of a photon. Therefore, these proteins represent signal converters that translate light into biological information. Researchers use this property to stimulate and study various biochemical processes conveniently and non-invasively by the application of light, an approach known as optogenetics. Here, we summarize the recent experimental progress on the family of blue light receptors using FAD (BLUF) receptors. Several BLUF photoreceptors modulate second messenger levels and thus represent highly interesting tools for optogenetic application. In order to activate a coupled effector protein, the flavin-binding pocket of the BLUF domain undergoes a subtle rearrangement of the hydrogen network upon blue light absorption. The hydrogen bond switch is facilitated by the ultrafast light-induced proton-coupled electron transfer (PCET) between a tyrosine and the flavin in less than a nanosecond and remains stable on a long enough timescale for biochemical reactions to take place. The cyclic nature of the photoinduced reaction makes BLUF domains powerful model systems to study protein/cofactor interaction, protein-modulated PCET and novel mechanisms of biological signalling. The ultrafast nature of the photoconversion as well as the subtle structural rearrangement requires sophisticated spectroscopic and molecular biological methods to study and understand this highly intriguing signalling process.

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Section: Photoinduced Electron Transfer In Bluf Domains: Redox Tuningmentioning

confidence: 99%

Molecular eyes: proteins that transform light into biological information

2013

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“…Expression conditions were first investigated at a TePhe concentration of 2.5 mM. As the BL21 (DE3) E. coli host used in this study is not auxotrophic for Phe, glyphosate ( N ‐phosphonomethyl)glycine), an inhibitor of 5‐enolpyruvyl‐shikimate‐3‐phosphate (EPSP) synthase, was added to the ex‐pression media to inhibit aromatic amino acid biosynthesis, as in established protocols for the incorporation of non‐canonical tyrosine analogues [30] . Glyphosate did not cause an appreciable dependence on exogenous phenylalanine in this strain, however, as protein yield did not decrease upon glyphosate addition (Table 1, entries 1 and 2).…”

Section: Resultsmentioning

confidence: 99%

“…Small‐scale screening of conditions for TePhe incorporation : Secondary cultures of BL21 (DE3) E. coli carrying the pET15b‐GB1 plasmid were grown in LB supplemented with 100 mg/mL ampicillin (60 mL culture per condition) at 37 °C with shaking until OD 600 reached 0.6‐0.8. Cells were harvested, washed once in PBS, and added to expression media (10 mL M9‐based media [30] with 100 mg/mL ampicillin, 2 mg/mL glyphosate, and appropriate amino acids) in either a tightly capped 15 mL starter culture tube for “low” aeration or a 50 mL Erlenmeyer flask for “normal” aeration, shaking at 200 rpm. Cells were then induced to express His 6 ‐GB1 using a final concentration of 1 mM IPTG under conditions indicated in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Incorporation of TePhe into Expressed Proteins is Minimally Perturbing

Nitz

2021

ChemBioChem

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Tellurium is a versatile heavy chalcogen with numerous applications in chemical biology, providing valuable probes in mass cytometry, fluorescence imaging and structural biology. L-Tellurienylalanine (TePhe) is an analogue of the proteinogenic amino acid L-phenylalanine (Phe) in which the phenyl side chain has been replaced by a 5-membered tellurophene moiety. High incorporation level of TePhe in expressed proteins at defined sites is expected to facilitate studies in proteomics, protein NMR spectroscopy, and structure elucidation. As a model we chose immunoglobulin-binding Protein G, B1 domain (GB1) to validate TePhe as a suitable structural analogue for Phe. We demonstrate that approximately 1 in 2 of all Phe sites within GB1 can be substituted with TePhe through expression in standard non-Phe-auxotrophic E. coli in Phe-deficient media containing glyphosate, an inhibitor of aromatic amino acid biosynthesis. The TePhe content of the GB1 sample can be further increased to 85 % through HPLC. Using NMR and CD spectroscopy, we confirm that the Phe-to-TePhe substitution has negligible impact on the global structure and stability of GB1.

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“…Thereby complete labeling of all corresponding nuclei in the protein - and cofactor, if provided through biosynthesis by the expression host - is achieved. In many cases, however, a more selective labeling is desired, in order to reduce the amount of couplings in NMR or to specifically assign a signal in an IR difference spectrum [ 27 , 28 ]. A common way to achieve a reduced, but controlled isotope labeling is to feed specific, but non-uniform labeled carbon sources, which lead to a preferential isotope enrichment in certain amino acids [ 29 – 31 ], or to employ other carbon breakdown and de novo amino acid synthesis pathways [ 32 ].…”

Section: Introductionmentioning

confidence: 99%

“…), which do not serve as intermediates for other amino acid biosynthesis pathways and are not likely to cause undesired scrambling of isotopes, are especially suitable in this approach [ 33 ]. Furthermore, isotope scrambling and dilution can be suppressed by providing substrates that lead to feedback inhibition of chosen biochemical pathways or by application of selective inhibitors [ 28 , 34 ]. Most of these methods require extensive optimization of the cultivation conditions due to the complexity of the therefore required media.…”

Section: Introductionmentioning

confidence: 99%

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

et al. 2013

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Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

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Side-chain specific isotopic labeling of proteins for infrared structural biology: The case of ring-D4-tyrosine isotope labeling of photoactive yellow protein

Cited by 8 publications

References 49 publications

Molecular eyes: proteins that transform light into biological information

Molecular eyes: proteins that transform light into biological information

Incorporation of TePhe into Expressed Proteins is Minimally Perturbing

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

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