How to change the oligomeric state of a circular protein assembly: switch from 11-subunit to 12-subunit TRAP suggests a general mechanism

Many critical cellular functions are performed by multisubunit circular protein oligomers whose internal geometry has evolved to meet functional requirements. The subunit number is arguably the most critical parameter of a circular protein assembly, affecting the internal and external diameters of the assembly and often impacting on the protein's function. Although accurate structural information has been obtained for several circular proteins, a lack of accurate information on alternative oligomeric states has prevented engineering such transitions. In this study we used the bacterial transcription regulator TRAP as a model system to investigate the features that define the oligomeric state of a circular protein and to question how the subunit number could be manipulated.We find that while Bacillus subtilis and Bacillus stearothermophilus TRAP form 11-subunit oligomers, the Bacillus halodurans TRAP exclusively forms 12-subunit assemblies. Significantly, the two states of TRAP are related by a simple rigid body rotation of individual subunits around inter-subunit axes. We tested if such a rotation could be induced by insertion or deletion mutations at the subunit interface. Using wild type 11-subunit TRAP, we demonstrate that removal of five C-terminal residues at the outer side of the inter-subunit axis or extension of an amino acid side chain at the opposite, inner side, increased the subunit number from 11 to 12. Our findings are supported by crystal structures of TRAP oligomers and by native mass spectrometry data.The subunit number of the TRAP oligomer can be manipulated by introducing deletion or addition mutations at the subunit interface. An analysis of available and emerging structural data on alternative oligomeric states indicates that the same principles may also apply to the subunit number of other circular assemblies suggesting that the deletion/addition approach could be used generally to engineer transitions between different oligomeric states

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products

A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further stud

Improving Predictions for Helium Emission Lines

We have combined the detailed He I recombination model of Smits with the collisional transitions of Sawey & Berrington in order to produce new accurate helium emissivities that include the effects of collisional excitation from both the 2 (3)S and 2 (1) S levels. We present a grid of emissivities for a range of temperature and densities along with analytical fits and error estimates. Fits accurate to within 1% are given for the emissivities of the brightest lines over a restricted range for estimates of primordial helium abundance. We characterize the analysis uncertainties associated with uncertainties in temperature, density, fitting functions, and input atomic data. We estimate that atomic data uncertainties alone may limit abundance estimates to an accuracy of 1.5%; systematic errors may be greater than this. This analysis uncertainty must be incorporated when attempting to make high accuracy estimates of the helium abundance. For example, in recent determinations of the primordial helium abundance, uncertainties in the input atomic data have been neglected.Comment: ApJ, accepte

Emerging evidence for CHFR as a cancer biomarker : from tumor biology to precision medicine

Novel insights in the biology of cancer have switched the paradigm of a "one-size-fits-all" cancer treatment to an individualized biology-driven treatment approach. In recent years, a diversity of biomarkers and targeted therapies has been discovered. Although these examples accentuate the promise of personalized cancer treatment, for most cancers and cancer subgroups no biomarkers and effective targeted therapy are available. The great majority of patients still receive unselected standard therapies with no use of their individual molecular characteristics. Better knowledge about the underlying tumor biology will lead the way toward personalized cancer treatment. In this review, we summarize the evidence for a promising cancer biomarker: checkpoint with forkhead and ring finger domains (CHFR). CHFR is a mitotic checkpoint and tumor suppressor gene, which is inactivated in a diverse group of solid malignancies, mostly by promoter CpG island methylation. CHFR inactivation has shown to be an indicator of poor prognosis and sensitivity to taxane-based chemotherapy. Here we summarize the current knowledge of altered CHFR expression in cancer, the impact on tumor biology and implications for personalized cancer treatment

Radio Emission from GRO J1655-40 during the 1994 Jet Ejection Episodes

We report multifrequency radio observations of GRO J1655-40 obtained with the Australia Telescope Compact Array, the Molonglo Observatory Synthesis Telescope and the Hartebeesthoek Radio Astronomy Observatory at the time of the major hard X-ray and radio outbursts in 1994 August-September. The radio emission reached levels of the order of a few Jy and was found to be linearly polarized by up to 10%, indicating a synchrotron origin. The light curves are in good agreement with those measured with the VLA, but our closer time sampling has revealed two new short-lived events and significant deviations from a simple exponential decay. The polarization data show that the magnetic field is well ordered and aligned at right angles to the radio jets for most of the monitoring period. The time evolution of the polarization cannot be explained solely in terms of a simple synchrotron bubble model, and we invoke a hybrid `core-lobe' model with a core which contributes both synchrotron and free-free emission and `lobes' which are classical synchrotron emitters.Comment: 36 pages, 5 tables, 9 figures; accepted for publication in Ap

Functional regeneration at the blood-biomaterial interface

The use of cardiovascular implants is commonplace in clinical practice. However, reproducing the key bioactive and adaptive properties of native cardiovascular tissues with an artificial replacement is highly challenging. Exciting new treatment strategies are under development to regenerate (parts of) cardiovascular tissues directly in situ using immunomodulatory biomaterials. Direct exposure to the bloodstream and hemodynamic loads is a particular challenge, given the risk of thrombosis and adverse remodeling that it brings. However, the blood is also a source of (immune) cells and proteins that dominantly contribute to functional tissue regeneration. This review explores the potential of the blood as a source for the complete or partial in situ regeneration of cardiovascular tissues, with a particular focus on the endothelium, being the natural blood-tissue barrier. We pinpoint the current scientific challenges to enable rational engineering and testing of blood-contacting implants to leverage the regenerative potential of the blood.</p

Gate-bias assisted charge injection in organic field-effect transistors

The charge injection barriers in organic field-effect transistors (OFETs) seem to be far less critical as compared to organic light-emitting diodes (OLEDs). Counter intuitively; we show that the origin is image-force lowering of the barrier due to the gate bias at the source contact; although the corresponding gate field is perpendicular to the channel current. In coplanar OFETs; injection barriers up to 1 eV can be surmounted by increasing the gate bias; enabling extraction of bulk transport parameters in this regime. For staggered transistors; however; the injection is gate-assisted only until the gate bias is screened by the accumulation channel opposite to the source contact. The gate-assisted injection is supported by two-dimensional numerical charge transport simulations that reproduce the gate-bias dependence of the contact resistance and the typical S-shaped output curves as observed for OFETs with high injection barriers.

Size of the Vela Pulsar's Radio Emission Region: 500 km

We use interstellar scattering of the Vela pulsar to determine the size of its emission region. From interferometric phase variations on short baselines, we find that radio-wave scattering broadens the source by 3.4+/-0.3 milliarcseconds along the major axis at position angle 81+/-3 degrees. The ratio of minor axis to major axis is 0.51+/-0.03. Comparison of angular and temporal broadening indicates that the scattering material lies in the Vela-X supernova remnant surrounding the pulsar. From the modulation of the pulsar's scintillation on very short baselines, we infer a size of 500 km for the pulsar's emission region. We suggest that radio-wave refraction within the pulsar's magnetosphere may plausibly explain this size.Comment: 14 pages, includes 2 figures. Also available at: http://charm.physics.ucsb.edu:80/people/cgwinn/cgwinn_group/cgwinn_group.htm