Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: <i>Escherichia coli</i> Ribonucleotide Reductase as a Paradigm

Lee, Wan-Kyu; Kasanmascheff, Müge; Huynh, Michael; Quartararo, Anthony J.; Costentin, Cyrille; Bejenke, Isabel; Nocera, Daniel G.; Bennati, Marina; Tommos, Cecilia; Stubbe, Jo Anne

doi:10.1021/acs.biochem.8b00160

Cited by 13 publications

(21 citation statements)

References 92 publications

(449 reference statements)

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“…Without tuning by its active-site environment (e.g., by control of protonation state), the DOPA radical (DOPA•) implicated by the crystallographic and LC-MS/MS data would not be expected to have a reduction potential sufficient for its generation of Cys• ( 31 , 32 ). Thus, it was important to verify the structure of the modified Tyr123 species in its functional, radical state.…”

Section: Resultsmentioning

confidence: 99%

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

Blaesi

Palowitch

Kai

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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SignificanceConversion of ribonucleotides to the 2′-deoxyribonucleotides required for DNA biosynthesis is catalyzed by ribonucleotide reductases (RNRs) via a free-radical mechanism. Known types of RNRs all depend on redox-active transition metals—manganese, iron, or cobalt—for radical initiation. Pathogenic bacteria are challenged by transition metal sequestration and infliction of oxidative stress by their hosts, and the deployment of multiple RNRs with different metal requirements and radical-initiating oxidants is a known bacterial countermeasure. A class I RNR from two bacterial pathogens completely lacks transition metals in its active state and uses a tyrosine-derived dihydroxyphenylalanine radical as its initiator, embodying a novel tactic to combat transition metal- and oxidant-mediated innate immunity and reinforcing bacterial RNRs as potential antibiotic targets.

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Section: Resultsmentioning

confidence: 99%

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

Blaesi

Palowitch

Kai

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…The systematic absence of experimentally determined values of Z has likely impeded a rigorous understanding of most chemical processes in which proteins are involved including aggregation and self‐assembly, ligand binding, catalysis, electron transfer, protein crystallization, analytical separation, and protein engineering . It is tempting to assume that the formal net charge of a protein predicted from generalized residue p K a values ( Z seq ) is so similar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about a protein's net charge.…”

Section: Mass Spectrometer‐yes Charge Spectrometer‐nomentioning

confidence: 99%

“…[1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering. [51][52][53][54][55][56] It is tempting to assume that the formal net chargeo faprotein predicted from generalized residue pK a values (Z seq )issosimilar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about ap rotein's net charge.…”

Section: Introductionmentioning

confidence: 99%

What Are We Missing by Not Measuring the Net Charge of Proteins?

Zahler

Shaw

2019

Chemistry A European J

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The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials—including contributions from tightly bound metal, solvent, buffer, and cosolvent ions—and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pKa. Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein—its mass—one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pKa values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔGz) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔGz contribute more to the redox potential? And what about metal binding itself? When an apo‐metalloprotein, bearing minimal net negative charge (e.g., Z=−2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool—the “protein charge ladder”—and used it to begin to answer these basic questions.

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“…As reference groups we have somewhat arbitrarily chosen those of Marina Bennati (Göttingen) [209][210][211], Robert Bittl (Berlin) [212][213][214], Dave Britt (UC Davis) [215][216][217], Jack Freed (Cornell) [218][219][220], Daniella Goldfarb (Weizmann) [221][222][223], Brian Hoffman (Northwestern) [224][225][226], Gunnar Jeschke (Zurich) [227][228][229], Chris Kay (London) [230,231], Yasuhiro Kobori (Kobe) [232][233][234], Wolfgang Lubitz (Mülheim/Ruhr) [235][236][237], Damian Murphy (Cardiff) [238][239][240], Thomas Prisner (Frankfurt/Main) [241][242][243], Christiane Timmel (Oxford) [244][245][246], Sabine van Doorslaer (Antwerp) [247][248][249], and Stefan Weber (Freiburg) [250][251][252].…”

Section: Pulse Endormentioning

confidence: 99%

“…Because of limited space for this review we will refrain from such a challenging endeavor. Instead, we have compiled a list of recent publications concerning EPR/PELDOR/DEER/DQC work since 2014/2015, using either nitroxides or Gd 3+ as spin labels; the respective references are: [6,[209][210][211][212][213][214][219][220][221][222][223]229,242,251,333,334,345,346,357,358,361,[374][375][376][377][378][379][380].…”

Section: Overview Of Studies On Nitroxide Spin-labeled Proteins and Dmentioning

confidence: 99%

Biomolecular EPR Meets NMR at High Magnetic Fields

et al. 2018

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In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

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Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

Cited by 13 publications

References 92 publications

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

What Are We Missing by Not Measuring the Net Charge of Proteins?

Biomolecular EPR Meets NMR at High Magnetic Fields

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