Letter to the Editor: 1H, 15N, and 13C chemical shift assignments of the resuscitation promoting factor domain of Rv1009 from Mycobacterium tuberculosis

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Volume of Hsp90 Protein-Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR

© 2016 American Chemical Society.Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein-ligand systems. Protein-ligand binding volumes were negative, suggesting that the protein-ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2

Improvements of the DANTE-Z sequence for band-selective excitation: application to multidimensional NMR spectroscopy

New developments of the DANTE-Z sequence (1) are presented. Particularly, improvements of the shape of the excitation profile are described. The easy implementation of DANTE-Z in the classic multidimensional homo- or heteronuclear experiments, and its numerous advantages (clean excitation profile, absence of phase gradient and of amplitude distorsions, no need of instrumental adjustments...) make this sequence the easier way to perform band- selective excitation in NMR spectroscopy. Most of these modifications have been checked on a sample of protein (toxin γ from Naja nigricollis (2)) dissolved in water

Band-filtering in multidimensional NMR

The advantages of band-filtering schemes, based on the selection of an inversion profile, over selective 90° excitation pulse are presented in case of multidimensional NMR spectroscopy. The performances of two "user-friendly" methods based on such a scheme, DANTE-Z and SPIN-PINGING, are discussed. In association with the use of pulse-width-modulated DANTE trains, these methods changes the tedious calibration of band-selective pulses into the calibration of a conventional π/2 "hard" pulse

Low-cost equilibrium unfolding of heme proteins using 2 μl samples

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure-function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b5, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed