NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium.

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions.

The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions.

Calmodulin mutations causing catecholaminergic polymorphic ventricular tachycardia confer opposing functional and biophysical molecular changes.

Calmodulin (CaM) is the central mediator of intracellular Ca(2+) signalling in cardiomyocytes, where it conveys the intricate Ca(2+) transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms.

Identification of a high-affinity network of secretagogin-binding proteins involved in vesicle secretion.

Secretagogin is a hexa EF-hand Ca(2+)-binding protein expressed in neuroendocrine, pancreatic endocrine and retinal cells. The protein has been noted for its expression in specific neuronal subtypes in the support of hierarchical organizing principles in the mammalian brain. Secretagogin has previously been found to interact with SNAP25 involved in Ca(2+)-induced exocytosis.

pK(a) values for the unfolded state under native conditions explain the pH-dependent stability of PGB1.

Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population.

Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus.

An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator Rex of S. aureus binds to this inverted repeat.

Integrated protein array screening and high throughput validation of 70 novel neural calmodulin-binding proteins.

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library.

Protein GB1 folding and assembly from structural elements.

Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy.

Molecular design of specific metal-binding peptide sequences from protein fragments: theory and experiment.

A novel strategy is presented for designing peptides with specific metal-ion chelation sites, based on linking computationally predicted ion-specific combinations of amino acid side chains coordinated at the vertices of the desired coordination polyhedron into a single polypeptide chain. With this aim, a series of computer programs have been written that 1) creates a structural combinatorial library containing Zi-(X)n-Zj sequences (n=0-14; Z: amino acid that binds the metal through the side chain; X: any amino acid) from the existing protein structures in the non-redundant Protein Data Bank; 2) merges these fragments into a single Z1-(X)n1 -Z2-(X)n2 -Z3-(X)n3 -..

Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex.

The transcription factor Rex has been implicated in regulation of the expression of genes important for fermentative growth and for growth under conditions of low oxygen tension in several Gram-positive bacteria. Rex senses the redox poise of the cell through changes in the NADH/NAD(+) ratio. The crystal structures of two essentially identical Rex proteins, from Thermus aquaticus and T.

Calmodulin binding to the polybasic C-termini of STIM proteins involved in store-operated calcium entry.

Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store-operated calcium entry in the cell. We show by isothermal titration calorimetry that calmodulin binds in a calcium-dependent manner to the polybasic C-termini of STIM1 and STIM2, a region critical for their translocation to the plasma membrane ( K D < or = 1 microM in calcium). HSQC NMR spectroscopy shows this interaction is in the fast exchange regime.

Zn2+ binding to human calbindin D(28k) and the role of histidine residues.

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events.

Intra- versus intermolecular interactions in monellin: contribution of surface charges to protein assembly.

The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments.

Salting the charged surface: pH and salt dependence of protein G B1 stability.

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.