Modeling the sequence dependence of differential antibody binding in the immune response to infectious disease.

Past studies have shown that incubation of human serum samples on high density peptide arrays followed by measurement of total antibody bound to each peptide sequence allows detection and discrimination of humoral immune responses to a variety of infectious diseases. This is true even though these arrays consist of peptides with near-random amino acid sequences that were not designed to mimic biological antigens. This "immunosignature" approach, is based on a statistical evaluation of the binding pattern for each sample but it ignores the information contained in the amino acid sequences that the antibodies are binding to. Here, similar array-based antibody profiles are instead used to train a neural network to model the sequence dependence of molecular recognition involved in the immune response of each sample. The binding profiles used resulted from incubating serum from 5 infectious disease cohorts (Hepatitis B and C, Dengue Fever, West Nile Virus and Chagas disease) and an uninfected cohort with 122,926 peptide sequences on an array. These sequences were selected quasi-randomly to represent an even but sparse sample of the entire possible combinatorial sequence space (~1012). This very sparse sampling of combinatorial sequence space was sufficient to capture a statistically accurate representation of the humoral immune response across the entire space. Processing array data using the neural network not only captures the disease-specific sequence-binding information but aggregates binding information with respect to sequence, removing sequence-independent noise and improving the accuracy of array-based classification of disease compared with the raw binding data. Because the neural network model is trained on all samples simultaneously, a highly condensed representation of the differential information between samples resides in the output layer of the model, and the column vectors from this layer can be used to represent each sample for classification or unsupervised clustering applications.

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

All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.

g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance.

Interpretation of magnetic resonance data in the context of structural and chemical biology requires prior knowledge of the g-tensor directions for paramagnetic metallo-cofactors with respect to the protein structural frame. Access to this information is often limited by the strict requirement of suitable protein crystals for single-crystal electron paramagnetic resonance (EPR) measurements or the reliance on protons (with ambiguous locations in crystal structures) near the paramagnetic metal site. Here we develop a novel pulsed EPR approach with selective 13Cß-cysteine labeling of model [2Fe-2S] proteins to help bypass these problems. Analysis of the 13Cß-cysteine hyperfine tensors reproduces the g-tensor of the Pseudomonas putida ISC-like [2Fe-2S] ferredoxin (FdxB). Its application to the hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus, for which the single-crystal EPR approach was not feasible, supports the best-fit g x-, g z-, and g y-tensor directions of the reduced cluster as nearly along Fe-Fe, S-S, and the cluster plane normal, respectively. These approximate principal directions of the reduced ARF g-tensor, explored by 13C pulsed EPR, are less skewed from the cluster molecular axes and are largely consistent with those previously determined by single-crystal EPR for the cytochrome bc1-associated, reduced Rieske [2Fe-2S] center. This suggests the approximate g-tensor directions are conserved across the phylogenetically and functionally divergent Rieske-type [2Fe-2S] proteins.

Glutamate 52-ß at the α/ß subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and ß subunits that form a transient, active α2ß2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/ß) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2ß2 created from X-ray structures of α and ß and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/ß interface (Glu350 and Glu52 in ß2 and Arg329 and Arg639 in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-ß2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F3Y122•) replacing the stable Tyr122• in WT-ß2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His6-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr356•-ß2), 0.5 eq of dCDP/F3Y122•, and formation of an α2ß2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2ß2 complex formed.

Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by 13Cß-Cysteine Labeling.

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4-n (n = 0, 1, 2) cluster by selective cysteine-13Cß isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cß hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2ß2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in ß2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/ß interface. The absence of an α2ß2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in ß2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type ß2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-ß2 and 2,3,5-F3Y122•-ß2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase.

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α2:ß2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122ß â†” [W48ß?] ↔ Y356ß â†” Y731α ↔ Y730α ↔ C439α). The mutant R411A(α) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in α2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active α2:ß2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-α2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns-µs time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 104 s-1 when comparing wild type to R411A-α2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer.

Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized α3Y model protein has allowed the first determination of formal reduction potentials (E°') for a Y residing within a protein (Berry, B. W.; Martínez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9739-9743). Using Schultz's technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into α3Y. The global protein properties of the resulting α3(3,5)F2Y, α3(2,3,5)F3Y, α3(2,3)F2Y and α3(2,3,6)F3Y variants are essentially identical to those of α3Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°'s of the very high-potential α3FnY proteins. E°'(pH 5.5; α3FnY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The FnY analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined α3FnY ΔE°'s to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz' = 11.8 MHz, whereas for H2, Tz' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes.

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122•) in one subunit (ß2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356•, Y731•, and Y730• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122• and Y356• has not been detected. Our recent report of Y356• formation in a kinetically and chemically competent fashion in the reaction of ß2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122•-ß2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122• and Y356•. Incubation of F3Y122•-ß2, Y731F-α2 (or Y730F-α2), CDP, and ATP at different temperatures (2-37 °C) provides ΔE°'(F3Y122•-Y356•) of 20 ± 10 mV at 25 °C. The pH dependence of the F3Y122• ⇆ Y356• interconversion (pH 6.8-8.0) reveals that the proton from Y356 is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356 and rapid freeze-quench EPR analysis of its reaction with Y731F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F2Y356• generation by the native Y122•. FnY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y122 and C439 is ∼200 meV uphill.

Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides.

Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the interactions of the protein with the quinones in the Q(A) and Q(B) sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the Q(B) site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the Q(A) and Q(B) sites. Disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the Q(A)⁻Q(B)⁻ biradical and competitive binding assays.

Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis.

Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.

The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.

Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe (13)C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group (13)C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of (13)C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9 ) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.

Tuning cofactor redox potentials: the 2-methoxy dihedral angle generates a redox potential difference of >160 mV between the primary (Q(A)) and secondary (Q(B)) quinones of the bacterial photosynthetic reaction center.

Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rhodobacter sphaeroides. (13)C hyperfine sublevel correlation measurements of the 2-methoxy in the semiquinone states, SQA and SQB, were compared with quantum mechanics calculations of the (13)C couplings as a function of the dihedral angle. X-ray structures support dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of ~180 mV. This is consistent with the failure of a ubiquinone analogue lacking the 2-methoxy to function as QB in mutant reaction centers with a ΔEm of ≈160-195 mV.

Conformational differences between the methoxy groups of QA and QB site ubisemiquinones in bacterial reaction centers: a key role for methoxy group orientation in modulating ubiquinone redox potential.

Ubiquinone is an almost universal, membrane-associated redox mediator. Its ability to accept either one or two electrons allows it to function in critical roles in biological electron transport. The redox properties of ubiquinone in vivo are determined by its environment in the binding sites of proteins and by the dihedral angle of each methoxy group relative to the ring plane. This is an attribute unique to ubiquinone among natural quinones and could account for its widespread function with many different redox complexes. In this work, we use the photosynthetic reaction center as a model system for understanding the role of methoxy conformations in determining the redox potential of the ubiquinone/semiquinone couple. Despite the abundance of X-ray crystal structures for the reaction center, quinone site resolution has thus far been too low to provide a reliable measure of the methoxy dihedral angles of the primary and secondary quinones, QA and QB. We performed 2D ESEEM (HYSCORE) on isolated reaction centers with ubiquinones (13)C-labeled at the headgroup methyl and methoxy substituents, and have measured the (13)C isotropic and anisotropic components of the hyperfine tensors. Hyperfine couplings were compared to those derived by DFT calculations as a function of methoxy torsional angle allowing estimation of the methoxy dihedral angles for the semiquinones in the QA and QB sites. Based on this analysis, the orientation of the 2-methoxy groups are distinct in the two sites, with QB more out of plane by 20-25°. This corresponds to an ≈50 meV larger electron affinity for the QB quinone, indicating a substantial contribution to the experimental difference in redox potentials (60-75 mV) of the two quinones. The methods developed here can be readily extended to ubiquinone-binding sites in other protein complexes.

Influence of Polar Mutations on the Electronic and Structural Properties of QA-· in Bacterial Reaction Centers.

Reaction centers from Rhodobacter sphaeroides with residue M265 mutated from isoleucine to threonine, serine, and asparagine (M265IT, M265IS, and M265IN, respectively) in the QA-· state are studied by high-resolution electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance spectroscopy methods to investigate the structural characteristics of these mutants influencing the redox properties of the QA site. All three mutants decrease the redox midpoint potential (Em) of QA by ∼0.1 V, yet the mechanism for this drop in Em is unclear. In this work, we examine (i) the hydrogen bonding interactions between QA-· and residues histidine M219 and alanine M260, (ii) the electron spin density distribution of the semiquinone, and (iii) the orientations of the ubiquinone methoxy substituents. 13C measurements show no significant contribution of methoxy dihedral angles to the observed decrease in Em for the QA mutants. Instead, 14N three-pulse ESEEM data suggest that electrostatic or hydrogen bond formation between the mutated M265 side chain and His-M219 Nδ may be involved in the observed lowering of the QA midpoint potential. For mutant M265IN, analysis of the proton hyperfine couplings reveals a weakened hydrogen bond network, resulting in an altered QA-· spin density distribution. The magnetic resonance study presented here is most consistent with an electrostatic or structural perturbation of the His-M219 Nδ hydrogen bond in these mutants as a mechanism for the ∼0.1 V decrease in QA Em.

Structure and biological evaluation of Caenorhabditis elegans CISD-1/mitoNEET, a KLP-17 tail domain homologue, supports attenuation of paraquat-induced oxidative stress through a p38 MAPK-mediated antioxidant defense response.

CISD-1/mitoNEET is an evolutionarily conserved outer mitochondrial membrane [2Fe-2S] protein that regulates mitochondrial function and morphology. The [2Fe-2S] clusters are redox reactive and shown to mediate oxidative stress in vitro and in vivo. However, there is limited research studying CISD-1/mitoNEET mediation of oxidative stress in response to environmental stressors. In this study, we have determined the X-ray crystal structure of Caenorhabditis elegans CISD-1/mitoNEET homologue and evaluated the mechanisms of oxidative stress resistance to the pro-oxidant paraquat in age-synchronized populations by generating C. elegans gain and loss of function CISD-1 models. The structure of the C. elegans CISD-1/mitoNEET soluble domain refined at 1.70-Å resolution uniquely shows a reversible disulfide linkage at the homo-dimeric interface and also represents the N-terminal tail domain for dimerization of the cognate kinesin motor protein KLP-17 involved in chromosome segregation dynamics and germline development of the nematode. Moreover, overexpression of CISD-1/mitoNEET in C. elegans has revealed beneficial effects on oxidative stress resistance against paraquat-induced reactive oxygen species generation, corroborated by increased activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade.

Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships.

A set of C43(DE3) and BL21(DE3) Escherichia coli host strains that are auxotrophic for various amino acids is briefly reviewed. These strains require the addition of a defined set of one or more amino acids in the growth medium, and have been specifically designed for overproduction of membrane or water-soluble proteins selectively labelled with stable isotopes, such as 2H, 13C and 15N. The strains described here are available for use and have been deposited into public strain banks. Although they cannot fully eliminate the possibility of isotope dilution and mixing, metabolic scrambling of the different amino acid types can be minimized through a careful consideration of the bacterial metabolic pathways. The use of a suitable auxotrophic expression host strain with an appropriately isotopically labelled growth medium ensures high levels of isotope labelling efficiency as well as selectivity for providing deeper insight into protein structure-function relationships.

Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex.

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and ß. Because of the transient subunit association, an atomic resolution structure of an active α2ß2 RNR complex has been elusive. We used a doubly substituted ß2, E52Q/(2,3,5)-trifluorotyrosine122-ß2, to trap wild-type α2 in a long-lived α2ß2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.