Kurt D. Berndt

Redox Potentials of Glutaredoxins and Other Thiol-Disulfide Oxidoreductases of the Thioredoxin Superfamily Determined by Direct Protein-Protein Redox Equilibria

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2C). Standard state redox potential (E°′) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E°′ resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E°′ values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coliglutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E°′ = −233 and −198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculatedE°′ values from a number of proteins ranging from −270 mV for E. coli Trx to −124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as −240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures.

A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.

A Proteinaceous Gene Regulatory Thermometer in Salmonella

Novel utilization of the coiled-coil motif is presented that enables TlpA, an autoregulatory repressor protein in Salmonella, to sense temperature shifts directly and thereby to modulate the extent of transcription repression. Salmonella cells shifted to higher temperatures, such as those encountered at host entry, showed derepressed tlpA activity. tlpA::lacZ fusions indicated that the promoter itself is insensitive to thermal shifts and that transcription control was exerted by the autorepressor TlpA only. In vitro studies with highly purified TlpA showed concentration and temperature dependence for both fully folded conformation and function, indicating that the thermosensing in TlpA is based on monomer-to-coiled-coil equilibrium.

The program ASNO for computer-supported collection of NOE upper distance constraints as input for protein structure determination

SummaryA new program, ASNO (‘ASsign NOes’), for computer-supported NOE cross-peak assignments is described. ASNO is used for structure refinement in several rounds of NOESY cross-peak assignments and 3D structure calculations, where the preliminary structures are used as a reference to resolve ambiguities in NOE assignments which are otherwise based on the chemical shifts available from the sequence-specific resonance assignments. The practical use of ASNO for proteins is illustrated with the structure determination of Dendrotoxin K from Dendroaspis polylepis polylepis.

The estrogen receptor α-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice; potential molecular mechanisms

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond.

The active site of Escherichia coli glutaredoxin‐3 (Grx3) consists of two redox active cysteine residues in the sequence ‐C11‐P‐Y‐C14‐H‐. The 1H NMR resonance of the cysteine thiol proton of Cys‐14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys‐11 thiolate, which is shown to have an abnormally low pK a value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys‐11 thiolate.

Thermal unfolding of small proteins with SH3 domain folding pattern

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two‐state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk‐, Itk‐, and Tec‐SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12–16 kJ/mol but show quite high melting temperatures.

Redox properties and evolution of human glutaredoxins

Glutaredoxins (Grxs) are glutathione‐dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol‐disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C‐X‐Y‐C active site sequence motif, the redox potentials of hGrx1 and hGrx2 were determined to be −232 and −221 mV, respectively, using a combination of redox buffers, protein–protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol−1 additional stability by linking the C‐terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be −317 mV and is thus expected to be present in all but the most reducing conditions in vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C‐G‐F‐S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin of vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins. Proteins 2007.

The estrogen receptor alpha-selective agonist PPT improves glucose tolerance in ob/ob mice; potential molecular mechanisms

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

DNA Binding Exerted by a Bacterial Gene Regulator with an Extensive Coiled-coil Domain

Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) α-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel, sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.