Genetically Encoded Protein Tyrosine Nitration in Mammalian Cells

Porter, Jason; Jang, Hyo Sang; Fossen, Elise M. Van; Nguyen, Duy; Willi, Taylor; Cooley, Richard B.; Mehl, Ryan A.

doi:10.1021/acschembio.9b00371

Cited by 27 publications

(61 citation statements)

References 33 publications

(53 reference statements)

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“…This is a significant change when considering P + BAis estimated to be ~70 meV below P* in energy in WT (Fig. 1B) (14,38,55). In fact, in a recently published study performed at a high level of theory, Tamura et al (22) indicate that P + BAis above P* in energy in WT RCs, which, if correct, would mean (1) that the P* → P + BA -→ P + HA -2-step process would not occur, and (2) that an ~100 meV energetic increase would be even more significant.…”

Section: Verification Of Ncaa Incorporation and Minimal Structural Perturbationmentioning

confidence: 97%

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Photosynthetic Reaction Center Variants Made via Genetic Code Expansion Show Tyr at M210 Tunes the Initial Electron Transfer Mechanism

Weaver

Lin²,

Faries³

et al. 2021

Preprint

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Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine close to an essential electron transfer component (M210) via its replacement with site-specific genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and x-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild-type. Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via an approximately 4 ps and 20 ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the 4 ps population P+HA- forms through a 2-step process P* –> P+BA– –> P+HA-, while in the 20 ps population it forms via a 1-step P* –> P+HA– superexchange mechanism. The percentage of P* population that decays via the superexchange route varies from approximately 25% to 45% among variants while in wild-type this percentage is approximately 15%. Increases in the P* population which decays via superexchange correlates with increases in free energy of the P+BA– intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an approximately 110 meV increase in the free energy of P+BA– along with a dramatic diminution of the 1030 nm transient absorption band indicative of P+BA– formation. Collectively this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.<br>

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Section: Verification Of Ncaa Incorporation and Minimal Structural Perturbationmentioning

confidence: 97%

“…S1.1 and S1.3-S1.4). We apply the same strategy used earlier (36) to a recently created amber suppression system (38) and genetically encode 3nitrotyrosine incorporation for the first time in R. sphaeroides (SI Secs. S1.1-S1.4).…”

Section: Verification Of Ncaa Incorporation and Minimal Structural Perturbationmentioning

confidence: 99%

Photosynthetic Reaction Center Variants Made via Genetic Code Expansion Show Tyr at M210 Tunes the Initial Electron Transfer Mechanism

Weaver

Lin²,

Faries³

et al. 2021

Preprint

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“…The most active and selective variants (32,41,82) had similar mutations in the active site (N311S and V366A, as well as W382T or W382V). Mb AcdRS 32 and 82 also had an additional active site mutation of C313A or C313G, respectively, and Mb AcdRS 82 also contained a non-active site mutation, L155V.…”

Section: Acd Synthetase Selectionmentioning

confidence: 99%

“…To identify a Mb PylRS/tRNACUA pair capable of site-specifically incorporating Acd into proteins in response to an amber stop codon (TAG) we screened a library of Mb PylRS variants in which five active-site residues were randomized to all 20 amino acids (N311, C313, V366, W382, G387) using a standard life/death selection in E. coli. 32,33 Following a round of positive selection in the presence of Acd and a round of negative selection against canonical amino acids, 96 colonies were assessed for their ability to suppress a TAG codon interrupted sfGFP gene (sfGFP-TAG150) in the presence of Acd and suppressor tRNACUA. The top 25 performing clones were sequenced and 13 unique RS clones were identified with highly similar active site sequences (Fig.…”

Section: Acd Synthetase Selectionmentioning

confidence: 99%

Genetic Encoding of a Highly Photostable, Long Lifetime Fluorescent Amino Acid for Imaging in Mammalian Cells

Jones

Robkis

Blizzard

et al. 2021

Preprint

Self Cite

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Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

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“…It has been proposed that the level of salivary tyrosine, classified in a group of aromatic amino acids (AAA), may be used to discriminate clinical manifestations of OLP. In vivo , tyrosine (Tyr), and more specifically its derivative, e.g., 3-nitrotyrosine (3-NT), undergoes free radical modifications, making it a promising biomarker for the intensification of oxidative/nitrosative stress (Figure 1) [17–19]. With regard to ongoing free radical stress observed through apparent decrease in the key antioxidant enzymes (GPx,SOD, and CAT), Tyr nitration can block the mechanism associated with their recompensation (restoring their original antioxidant functions).…”

Section: Introductionmentioning

confidence: 99%

The Relationship between the Concentration of Salivary Tyrosine and Antioxidants in Patients with Oral Lichen Planus

Darczuk

Krzyściak

Bystrowska

et al. 2019

Oxidative Medicine and Cellular Longevity

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The diagnosis of oral lichen planus (OLP) is based on clinical examination and histopathological criteria. Noninvasive diagnostics of saliva may be considered as a confirmation of OLP diagnosis and a potential alternative to an invasive method. The objective of the present study was to evaluate the relationship between the level of tyrosine (Tyr) as well as antioxidants like uric acid (UA) and glutathione peroxidase (GPx) activity in the saliva of patients with OLP in comparison with the control group (healthy subjects without any oral changes). A total of 40 patients with OLP and 40 healthy volunteers were selected for the study based on the modified WHO diagnostic (clinical and histopathological) criteria. High-performance liquid chromatography (HPLC) was performed for Tyr concentration, while GPx activity and uric acid levels were determined by a colorimetric method. The concentrations of Tyr, UA, and GPx activity were statistically lowered in OLP patients compared to the control group. All examined parameters correlated strongly and positively with each other. Mean values of salivary UA concentrations differed between the groups of OLP patients (reticular and erosive forms) and controls (206.66 vs. 196.54 vs. 218.49 μmol/L, respectively, p = 0.001). A similar trend was demonstrated in salivary Tyr concentration which differed statistically between the study and control groups (0.08 vs. 0.07 vs. 0.13 μmol/L, respectively, p = 0.001). Determining of a relationship between the concentrations of Tyr, UA, and GPx activity may be useful in the prognosis of OLP. The HPLC method may be employed, as an additional noninvasive diagnostic procedure to screen OLP patients, during the routine diagnostics of salivary biochemical parameters such as aromatic amino acids.

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Genetically Encoded Protein Tyrosine Nitration in Mammalian Cells

Cited by 27 publications

References 33 publications

Photosynthetic Reaction Center Variants Made via Genetic Code Expansion Show Tyr at M210 Tunes the Initial Electron Transfer Mechanism

Photosynthetic Reaction Center Variants Made via Genetic Code Expansion Show Tyr at M210 Tunes the Initial Electron Transfer Mechanism

Genetic Encoding of a Highly Photostable, Long Lifetime Fluorescent Amino Acid for Imaging in Mammalian Cells

The Relationship between the Concentration of Salivary Tyrosine and Antioxidants in Patients with Oral Lichen Planus

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