Jeffrey L. Urbauer

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of 13C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of 13C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-13C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally 13C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.

Solution structure and stability of the anti-sigma factor AsiA: Implications for novel functions

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli, σ70. AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix–turn–helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen/deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to σ70. The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to σ70, where an AsiA protomer is displaced from the dimer via the interaction of σ70 with the same residues in AsiA that constitute the dimer interface.

Interaction of T4 AsiA with its Target Sites in the RNA Polymerase σ70 Subunit Leads to Distinct and Opposite Effects on Transcription

Bacteriophage T4 AsiA is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of Escherichia coli RNA polymerase holoenzyme (Esigma(70)) and preventing promoter complex formation on most E.coli and early T4 promoters. In addition, Esigma(70)AsiA, but not Esigma(70), is a substrate of transcription activation by T4-encoded DNA-binding protein MotA, a co-activator of transcription from middle viral promoters. The molecular determinants of sigma(70)-AsiA interaction necessary for transcription inhibition reside in the sigma(70) conserved region 4.2, which recognizes the -35 promoter consensus element. The molecular determinants of sigma(70)-AsiA interaction necessary for MotA-dependent transcription activation have not been identified. Here, we show that in the absence of sigma(70) region 4.2, AsiA interacts with sigma(70) conserved region 4.1 and activates transcription in a MotA-independent manner. Further, we show that the AsiA dimer must dissociate to interact with either region 4.2 or region 4.1 of sigma(70). We propose that MotA may co-activate transcription by restricting AsiA binding to sigma(70) region 4.1.

Proton-Induced Reactivity of NO– from a {CoNO}8 Complex

Research on the one-electron reduced analogue of NO, namely nitroxyl (HNO/NO–), has revealed distinguishing properties regarding its utility as a therapeutic. However, the fleeting nature of HNO requires the design of donor molecules. Metal nitrosyl (MNO) complexes could serve as potential HNO donors. The synthesis, spectroscopic/structural characterization, and HNO donor properties of a {CoNO}8 complex in a pyrrole/imine ligand frame are reported. The {CoNO}8 complex [Co(LN4PhCl)(NO)] (1) does not react with established HNO targets such as FeIII hemes or Ph3P. However, in the presence of stoichiometric H+1 behaves as an HNO donor. Complex 1 readily reacts with [Fe(TPP)Cl] or Ph3P to afford the {FeNO}7 porphyrin or Ph3P=O/Ph3P=NH, respectively. In the absence of an HNO target, the {Co(NO)2}10 dinitrosyl (3) is the end product. Complex 1 also reacts with O2 to yield the corresponding CoIII-η1-ONO2 (2) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}8 with biotargets such as FeIII-porphyrins.

Resonance assignment strategies for the analysis of NMR spectra of proteins.

Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the1H resonances based on 2D, homonuclear1H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews the evolution of strategies for the assignment of resonances of proteins.

Determinants of Affinity and Activity of the Anti-Sigma Factor AsiA †

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the sigma(70) subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-sigma(70) interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomer-protomer affinity in AsiA homodimers, to monomeric AsiA affinity for sigma(70), and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements were used to assess affinity and function of site-specific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer, V14, I17, and I40, resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiA-dependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation.

Localized production of human E-cadherin-derived first repeat in Escherichia coli

E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues. In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli. Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis. The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column. Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1. Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded. The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.

House fly cytochrome b5 exhibits kinetically trapped hemin and selectivity in hemin binding.

We report that cytochrome b(5) (cyt b(5)) from Musca domestica (house fly) is more thermally stable than all other microsomal (Mc) cytochromes b(5) that have been examined to date. It also exhibits a much higher barrier to equilibration of the two isomeric forms of the protein, which differ by a 180 degrees rotation about the alpha-gamma-meso axis of hemin (ferric heme). In fact, hemin is kinetically trapped in a nearly statistical 1.2:1 ratio of rotational forms in freshly expressed protein. The equilibrium ratio (5.5:1) is established only upon incubation at temperatures above 37 degrees C. House fly Mc cyt b(5) is only the second b-hemoprotein that has been shown to exhibit kinetically trapped hemin at room temperature or above, the first being cyt b(5) from the outer membrane of rat liver mitochondria (rat OM cyt b(5)). Finally, we show that the small excess of one orientational isomer over the other in freshly expressed protein results from selective binding of hemin by the apoprotein, a phenomenon that has not heretofore been established for any apocyt b(5).

Effects of deuterium on the kinetics of beef heart mitochondrial ATPase

A study was done examining the steady-state kinetics of F1-catalyzed ATP and ITP hydrolyses in the presence or absence of D2O as a function of temperature. The steady-state kinetic parameters kcat and kcat/Km were obtained. For ATP hydrolysis, kcat/Km was independent of temperature in the presence or absence of D2O, while kcat/Km for ITP hydrolysis increased in both cases. The relative magnitudes of change of kcat and kcat/Km in the presence and absence of D2O over the temperature range studied were much different for the cases of ATP and ITP hydrolysis. A normal isotope effect was observed in plots of kcat H2O/kcat D2O versus temperature for ATP hydrolysis, which increased then leveled off as temperature increased. An inverse isotope effect at low temperatures changed to a normal isotope effect and increased dramatically as temperature increased during ITP hydrolysis. The results are discussed in terms of the nature and location of the rate-limiting steps in the reaction mechanisms.

Membranous adenylyl cyclase 1 activation is regulated by oxidation of N- and C-terminal methionine residues in calmodulin

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca(2+)-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress. We examined how oxidation by H2O2 of Met in CaM regulates CaM activation of AC1. We employed a series of thirteen mutant CaM proteins never assessed before in a single study, where leucine is substituted for Met, in order to analyze the effects of oxidation of specific Met. CaM activation of AC1 is regulated by oxidation of all of the C-terminal Met in CaM, and by two N-terminal Met, M36 and M51. CaM with all Met oxidized is unable to activate AC1. Activity is fully restored by the combined catalytic activities of methionine sulfoxide reductases A and B (MsrA and B), which catalyze reduction of the (S)- and (R)-MetSO stereoisomers. A small change in secondary structure is observed in wild-type CaM upon oxidation of all nine Met, but no significant secondary structure changes occur in the mutant proteins when Met residues are oxidized by H2O2, suggesting that localized polarity, flexibility and structural changes promote the functional changes accompanying oxidation. The results signify that AC1 catalytic activity can be delicately adjusted by mediating CaM activation of AC1 by reversible Met oxidation in CaM. The results are important for memory, learning and possible therapeutic routes for regulating AC1.