Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates

The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence

E. coli Histidine Triad Nucleotide Binding Protein 1 (ecHinT) Is a Catalytic Regulator of D-Alanine Dehydrogenase (DadA) Activity In Vivo

Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both Gram-negative and Gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli

Harvard Personal Genome Project: lessons from participatory public research

Background: Since its initiation in 2005, the Harvard Personal Genome Project has enrolled thousands of volunteers interested in publicly sharing their genome, health and trait data. Because these data are highly identifiable, we use an ‘open consent’ framework that purposefully excludes promises about privacy and requires participants to demonstrate comprehension prior to enrollment. Discussion Our model of non-anonymous, public genomes has led us to a highly participatory model of researcher-participant communication and interaction. The participants, who are highly committed volunteers, self-pursue and donate research-relevant datasets, and are actively engaged in conversations with both our staff and other Personal Genome Project participants. We have quantitatively assessed these communications and donations, and report our experiences with returning research-grade whole genome data to participants. We also observe some of the community growth and discussion that has occurred related to our project. Summary We find that public non-anonymous data is valuable and leads to a participatory research model, which we encourage others to consider. The implementation of this model is greatly facilitated by web-based tools and methods and participant education. Project results are long-term proactive participant involvement and the growth of a community that benefits both researchers and participants

Image of the Energy Gap Anisotropy in the Vibrational Spectum of a High Temperature Superconductor

We present a new method of determining the anisotropy of the gap function in layered high-Tc superconductors. Careful inelastic neutron scattering measurements at low temperature of the phonon dispersion curves in the (100) direction in La_(1.85)Sr_(.15)CuO_4 would determine whether the gap is predominately s-wave or d-wave. We also propose an experiment to determine the gap at each point on a quasi-two-dimensional Fermi surface.Comment: 12 pages + 2 figures (included

Entropy and Correlators in Quantum Field Theory

It is well known that loss of information about a system, for some observer, leads to an increase in entropy as perceived by this observer. We use this to propose an alternative approach to decoherence in quantum field theory in which the machinery of renormalisation can systematically be implemented: neglecting observationally inaccessible correlators will give rise to an increase in entropy of the system. As an example we calculate the entropy of a general Gaussian state and, assuming the observer's ability to probe this information experimentally, we also calculate the correction to the Gaussian entropy for two specific non-Gaussian states.Comment: 23 pages, 9 figure

High energy parton-parton amplitudes from lattice QCD and the stochastic vacuum model

Making use of the gluon gauge-invariant two-point correlation function, recently determined by numerical simulation on the lattice in the quenched approximation and the stochastic vacuum model, we calculate the elementary (parton-parton) amplitudes in both impact-parameter and momentum transfer spaces. The results are compared with those obtained from the Kr\"{a}mer and Dosch ansatz for the correlators. Our main conclusion is that the divergences in the correlations functions suggested by the lattice calculations do not affect substantially the elementary amplitudes. Phenomenological and semiempirical information presently available on elementary amplitudes is also referred to and is critically discussed in connection with some theoretical issues.Comment: Text with 11 pages in LaTeX (twocolumn form), 10 figures in PostScript (psfig.tex used). Replaced with changes, Fig.1 modified, two references added, some points clarified, various typos corrected. Version to appear in Phys. Rev.

Comment on "Periodic Phase Synchronization in Coupled Chaotic Oscillators"

2 pages.-- PACS numbers: 05.45.Xt, 05.45.Pq.-- Final full-text version of the paper available at: http://dx.doi.org/10.1103/PhysRevE.73.038201.Kye et al. [Phys. Rev. E 68, 025201(R) (2003)] have recently claimed that, before the onset of Chaotic Phase Synchronization in coupled phase coherent oscillators, there exists a temporally coherent state called Periodic Phase Synchronization (PPS). Here we give evidence that some of their numerical calculations are flawed, while we provide theoretical arguments that indicate that PPS is not to be expected generically in this type of systems.This work was supported by MEC (Spain) and FEDER under Grant Nos. BFM2001-0341-C02-02, FIS2004-00953 (CONOCE2), and FIS2004-05073-C04-03.http://dx.doi.org/10.1103/PhysRevE.73.03820