Synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments.

This work reports the first synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilizing large α-helical membrane proteins in samples for [(15)N-(1)H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible.

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions.

¹H, ¹⁵N, and ¹³C backbone chemical shift assignment of titin domains A59-A60 and A60 alone.

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains.

Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release.

Unlabelled: Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority.

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid.

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold.