Oxidation State-dependent Protein-Protein Interactions in Disulfide Cascades

Abstract

Bacterial growth and pathogenicity depend on the correct formation of disulfide bonds, a process controlled by the Dsb system in the periplasm of Gram-negative bacteria. Proteins with a thioredoxin fold play a central role in this process. A general feature of thiol-disulfide exchange reactions is the need to avoid a long lived product complex between protein partners. We use a multidisciplinary approach, involving NMR, x-ray crystallography, surface plasmon resonance, mutagenesis, and in vivo experiments, to investigate the interaction between the two soluble domains of the transmembrane reductant conductor DsbD. Our results show oxidation state-dependent affinities between these two domains. These observations have implications for the interactions of the ubiquitous thioredoxin-like proteins with their substrates, provide insight into the key role played by a unique redox partner with an immunoglobulin fold, and are of general importance for oxidative protein-folding pathways in all organisms.

Direct and Indirect Involvement of MicroRNA-499 in Clinical and Experimental Cardiomyopathy

“…Among the down-regulated primarily affected mRNAs were 31 identified by TargetScan6 as potential direct miR-499 targets, 4 of which had been down-regulated in Gαq transgenic hearts (Figure 3c; Online Table IV) and 7 of which had been downregulated in human cardiomyopathy (Figure 3c, asterisks ). We 7, 20 and others 21, 22 have demonstrated the superiority of RNA-sequencing over microarray and RT-qPCR methods for precision, dynamic range and high-throughput characterization. Nonetheless, we validated three of these mRNAs using TaqMan RT-qPCR (Figure 3d).…”

Unconventional Targeting of a Thiol Peroxidase to the Mitochondrial Intermembrane Space Facilitates Oxidative Protein Folding

“…After 1 hr at room temperature (RT), 30 ng of each protein per reaction was incubated together for specific time points. Proteins were precipitated with 5% TCA, and AMS labeling was performed in buffer containing 50 mM Tris (pH 7.5), 3% w/v SDS, 3 mM EDTA, and 15 mM AMS for 30 min at 30°C and 30 min at 37°C (adapted from Mavridou et al., 2011). Proteins were run on non-reducing SDS-PAGE gels and visualized using Coomassie.…”

The interplay between the disulfide bond formation pathway and cytochrome c maturation in Escherichia coli

“…Growth media were prepared as described previously [30]. 10 mM nitrate (or 5 mM nitrite in the case of expression of endogenous cytochromes) was used as terminal electron acceptor.…”