Cytochrome cd1 nitrite reductases (cd 1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd 1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd 1NiR from Marinobacter hydrocarbonoclasticus (Mhcd 1) depends on the presence of its physiological redox partner, cytochrome c 552 (cyt c 552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd 1 and cyt c 552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd 1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd 1 // cyt c 552 and Ag // SAM // cyt c 552 // Mhcd 1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c 552 retains its native properties, while the redox potential of apparently intact Mhcd 1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd 1, reinforcing the idea that subtle and very specific interactions between Mhcd 1 and cyt c 552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd 1.

Muscle fatigue affects knee position sense, and the deleterious effect is not different depending upon the muscle group fatigued.

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.transcriptional activation | NMR | negative stain electron microscopy | protein-DNA complex | protein-protein complex C ellular responses to extracellular signals depend on a complex array of protein-protein interactions, including signaling cascades within the cytoplasm involving different types of posttranslational modification, ultimately leading to the activation of factors that mediate transcription of the appropriate response genes. One of the best-understood signaling molecules is NF-κB, a family of inducible transcription factors that is present in almost all cell types in higher eukaryotes. Its activation through various stimuli elicits variable responses, leading to transcription of target genes necessary for the immune response, inflammation, cellular growth, differentiation, cell adhesion, and cell survival (1). In mammals, the NF-κB family comprises a set of homo-or heterodimers of five monomer units, termed RelA (also known as p65), RelB, c-Rel, p50, and p52. The most abundant and bestknown NF-κB is the heterodimer of RelA and p50, present in the cytoplasm of resting cells in complex with its inhibitor IκBα, which masks the nuclear localization signal (NLS) of RelA. Upon receipt of a stimulus, a signaling cascade results in the phosphorylation, ubiquitination, and degradation of the bound IκBα; the RelA NLS is released; and the NF-κB enters the nucleus. Signal-responsive genes are regulated by the binding of NF-κB to the appropriate promoter. The activated NF-κB response is terminated through various mechanisms, including resynthesis of IκBα; the corresponding gene, nfkbia, is a strongly induced target gene of NF-κB. Newly synthesized IκBα enters the nucleus, recombines with the DNA-bound NF-κB, and the complex reverts to the resting state in the cytoplasm. The newly synthesized IκBα acts as an important component of the negative feedback loop that is one of the mechanisms for termination of NF-κB activation (2).A number of studies have illuminated the structural basis for the protein-protein interactions that NF-κB undergoes during the activation process. The X-ray crystal structure of the complex of RelA-p50 with a cognate DNA ...

NF-jB 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-jB 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-jB monomers p65 and p50 (72 kDa) prohibits the straightforward use of tripleresonance 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-jB 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-jB in the cell.

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 C α/β , and H N 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. K E Y W O R D S{ 1 H} -15 N NOE, NFκB1, p50 dimerization domain, protein dynamics, R 1 /R 2 relaxation