Comparison of backbone dynamics of the p50 dimerization domain of NFκB in the homodimeric transcription factor NFκB1 and in its heterodimeric complex with RelA (p65).

The nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) transcription factors play a critical role in human immune response. The family includes homodimers and heterodimers of five component proteins, which mediate different transcriptional responses and bind preferentially to different DNA sequences. Crystal structures of DNA complexes show that the dimers of the Rel-homology regions are structurally very similar. Differing DNA sequence preference together with structural similarity suggests that the dimers may differ in their dynamics. In this study, we present the first near-complete 15 N, 13 Cα/ß , and HN backbone resonance assignments of two dimers of the dimerization domain (DD) of the NFκB1 (p50) protein (residues 241-351): the homodimer of two p50 domains and a heterodimer of the p50 DD with the p65 DD. As expected, the two dimers behave very similarly, with chemical shift differences between them largely concentrated in the dimer interface and attributable to specific differences in the amino acid sequences of p50 and p65. A comparison of the picosecond-nanosecond dynamics of the homo- and heterodimers also shows that the environment of p50 is similar, with an overall slightly reduced correlation time for the homodimer compared to the heterodimer, consistent with its slightly smaller molecular weight. These results demonstrate that NMR spectroscopy can be used to explore subtle changes in structure and dynamics that have the potential to give insights into differences in specificity that can be exploited in the design of new therapeutic agents.

Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα.

The transcription factor NF-κB is used in many systems for the transduction of extracellular signals into the expression of signal-responsive genes. Published structural data explain the activation of NF-κB through degradation of its dedicated inhibitor IκBα, but the mechanism by which NF-κB-mediated signaling is turned off by its removal from the DNA in the presence of newly synthesized IκBα (termed stripping) is unknown. Previous kinetic studies showed that IκBα accelerates NF-κB dissociation from DNA, and a transient ternary complex between NF-κB, its cognate DNA sequence, and IκBα was observed. Here we structurally characterize the >100-kDa ternary complex by NMR and negative stain EM and show a modeled structure that is consistent with the measurements. These data provide a structural basis for previously unidentified insights into the molecular mechanism of stripping.

NMR characterization of a 72 kDa transcription factor using differential isotopic labeling.

NF-κB is a major transcription factor that mediates a number of cellular signaling pathways. Crystal structure analysis gives an incomplete picture of the behavior of the protein, particularly in the free state; free monomers or dimers of NF-κB have never been crystallized. NMR analysis gives insights into the structure and dynamics of the protein in solution, but a necessary first step is the assignment of resonances. The size of the heterodimer of the Rel homology regions of the NF-κB monomers p65 and p50 (72 kDa) prohibits the straightforward use of triple-resonance spectroscopy to obtain the assignments. However, the dynamic nature of the free heterodimer, in particular the independence of the DNA-binding and dimerization domains of each monomer, allows the assignments made on differentially labeled smaller domains to be mapped successfully onto the spectrum of the larger full-length RHR. Problematic areas such as the p65 nuclear localization sequence, which is disordered in the free protein, can be approached by residue-specific labeling and comparison with previously-published spectra of a short peptide with the same sequence. Overall, this NMR analysis of NF-κB has given valuable insights into the highly dynamic nature of the free state, which is likely to play an important role in the functional cycle of NF-κB in the cell.

SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1.

Electron transfer between multihaem cytochromes c3 from Desulfovibrio africanus.

The tetrahaem type I cytochromes c3 from Desulfovibrionaceae shuttle electrons from a periplasmic hydrogenase to transmembrane electron transfer complexes. In D. africanus, it is believed that the electrons are received by another tetrahaem cytochrome c3, denoted type II, which is associated with the membrane complex. Thermodynamic measurements show that the type I cytochrome c3 has the potential to transfer two electrons at a time. This study uses two-dimensional NMR to investigate the exchange of electrons between type I and type II cytochromes c3 at equilibrium in intermediate stages of oxidation. The results indicate that the two proteins are physiological partners but that only single-electron transfers occur in solution.

Relative importance of driving force and electrostatic interactions in the reduction of multihaem cytochromes by small molecules.

Multihaem cytochromes are essential to the energetics of organisms capable of bioremediation and energy production. The haems in several of these cytochromes have been discriminated thermodynamically and their individual rates of reduction by small electron donors were characterized. The kinetic characterization of individual haems used the Marcus theory of electron transfer and assumed that the rates of reduction of each haem by sodium dithionite depend only on the driving force, while electrostatic interactions were neglected. To determine the relative importance of these factors in controlling the rates, we studied the effect of ionic strength on the redox potential and the rate of reduction by dithionite of native Methylophilus methylotrophus cytochrome c″ and three mutants at different pH values. We found that the main factor determining the rate is the driving force and that Marcus theory describes this satisfactorily. This validates the method of the simultaneous fitting of kinetic and thermodynamic data in multihaem cytochromes and opens the way for further investigation into the mechanisms of these proteins.

Highly selective ligand binding by Methylophilus methylotrophus cytochrome c''.

Cytochrome c'' (cyt c'') from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c'' having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c''. Binding of CO, as well as CN(-), on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c'' for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe(2+)-NO adduct by stopped-flow and resonance Raman spectroscopy, respectively.

The effect of fatigue on knee position sense is not dependent upon the muscle group fatigued.

INTRODUCTION: Muscle fatigue could have a greater impact on position sense when antagonists of the movement are fatigued. Hence, this study aimed to compare the effects of antagonist and agonist exercise-induced muscle fatigue on knee joint position sense. METHODS: This within-subjects repeated-measures study included 40 subjects. Knee position sense and muscle strength were measured before and after two exercise protocols consisting of 30 consecutive maximal concentric/eccentric contractions of the knee extensors or flexors on the isokinetic dynamometer at an angular velocity of 180°/s (3.14 rad/s). RESULTS: Both exercise protocols increased the absolute angular error (F(1.78) = 39.89, P < 0.001; knee extensors protocol from 2.0 ± 1.3° to 3.5 ± 2.0°, knee flexors protocol from 2.1 ± 1.2° to 3.7 ± 2.2°), and no differences were detected between protocols (F(1.78) = 0.034, P = 0.855). No changes were observed in the relative angular error. CONCLUSIONS: Muscle fatigue affects knee position sense, and the deleterious effect is not different depending upon the muscle group fatigued.