Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.     

Peptide analysis: solid phase extraction-elution on diamond combined with atmospheric pressure matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) has been an indispensable ion generation technique for mass spectrometric analysis of biopolymers such as intact proteins and protein digests operated at atmospheric pressure. Since its advent in 1998, atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) quickly became a popular alternative for the analysis of peptides. Although AP-MALDI sources typically share the same vacuum interface and ion transmission hardware with ESI, it is generally found that ESI is superior in detection sensitivity. Here we present a method based on solid phase extraction and elution with surface-functionalized diamond nanocrystals (which we previously referred to as "SPEED") that not only streamlines AP-MALDI mass spectrometric analyses of peptides and other small biomolecules under typical operational conditions but also outruns ESI in ultimate detectable concentration by at least one order of magnitude.     

Bonding and Stability of Hybrid Diamond/Nanotube Structures

Geometrical considerations combined with detailed atomic modeling are used to define general classes of diamond/carbon nanotube interface structures with low residual stresses and no unsatisfied bonding. Chemically and mechanically robust interfaces are predicted, supporting recent experimental studies in which structures of this type were proposed.     

Properties of Polycrystalline Diamond: Multiscale Modeling Approach

Abstract

Two modeling techniques to characterize fracture behavior of polycrystalline diamond films are discussed. The first technique is a multiscale modeling method in which first-principles local density approximation calculations on selected structures are combined with an analytic mesoscale model to obtain energies and cleavage fracture energies for symmetric ?001? tilt grain boundaries (GBs) over the entire misorientation range. The second technique is large-scale atomistic simulation of the dynamics of failure in notched polycrystalline diamond samples under an applied strain. Electronic characteristics of selected ?001? symmetrical tilt GBs calculated with a semiempirical tight-binding Hamiltonian are also presented, and the possible role of graphitic defects on field emission from polycrystalline diamond is briefly discussed.     

Nearest neighbour considerations in Stillinger-Weber type potentials for diamond

Results of a preliminary investigation into the effect of varying the interaction cutoff on the bulk properties of diamond using a Stillinger-Weber (SW) type potential for C (Diamond) are presented. The interaction cutoff is varied over a range that includes and excludes the second-nearest neighbours. Whilst the original SW potential for silicon only included first-nearest neighbours inside the interaction cut-off, subsequent parameterizations for carbon (diamond) have also included second-nearest neighbours. Elastic and vibration properties of diamond were calculated over a range of cutoff distances used and the results show that certain lattice properties exhibit an approximately linear dependence on the interaction cut-off.     

Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid

In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.     

A chemical-genetic approach to elucidate protein kinase function <Emphasis Type="Italic">in planta</Emphasis>

The major objective in protein kinase research is the identification of the biological process, in which an individual enzyme is integrated. Protein kinase-mediated signalling is thereby often addressed by single knock-out mutation- or co-suppression-based reverse genetics approaches. If a protein kinase of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining ATP-analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant calcium-dependent protein kinase (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with ATP as substrate, but were able to use ATP analogues as phosphate donors or as kinase inhibitors. For functional characterization in planta, we have substituted an Arabidopsis thaliana mutant line of AtCPK1 with the respective as-variant under the native CPK1 promoter. Seedlings of Arabidopsis wild type and AtCPK1 as-lines were treated with the ATP analogue inhibitor 1-NA-PP1 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and AtCPK1 as-plants before and after inhibitor treatment revealed differential CPK1-dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.