Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet

Effect of Phosphate on the Kinetics and Specificity of Glycation of Protein

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffiner os rder to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues ino r near the activsei te. In contrast, in the cationic buffers, 3-(N-morpholino)propanesulfonic acid and 3-(N-tris(hydroxymethyl)rnethylamino)- 2-hydroxypropanesulfonica cid, the kineticso f glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNase was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increasegdly cation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effeocnt st he glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establishth at buffering ions or ligands can exert significant effects on the kinetics ands pecificity of glycation of proteins

Nonthermal Supermassive Dark Matter

We discuss several cosmological production mechanisms for nonthermal supermassive dark matter and argue that dark matter may be elementary particles of mass much greater than the weak scale. Searches for dark matter should not be limited to weakly interacting particles with mass of the order of the weak scale, but should extend into the supermassive range as well.Comment: 11 page LaTeX file. No major changes. Version accepted by PR

Modelling predictive gender- and gestation-specific weight reference centiles for preterm infants using a population-based cohort study

We aimed to model longitudinal data to create predictive growth charts for weight in preterm infants from birth till discharge, that took into account the differing growth rates post-birth when compared to in-utero growth and therefore was more representative of the data than the UK1990 reference charts. Data from birth until discharge (or death), was collected and rigorously cleaned for all infants born at <32 weeks of gestation over a 4-year period. Means and standard deviations from the UK1990 reference charts were used to compute standard deviation scores (SDS) for our cohort. 2/3rd of the data was randomly selected and used to create gestation and gender-specific predictive weight centile lines through novel application of mixed modelling methods. The remaining 1/3rd of the data was used to test model fit by comparing expected vs actual weights for the new model with those predicted by the UK1990 model. Data from 1,510 preterm infants was analysed. 1067 of these were used to produce the predictive model. Weekly SDS were significantly lower than predicted throughout hospital stay for all gestation groups when compared with UK1990 data. The test data (n = 539) fitted the new centile lines substantially better than those modelled by the UK1990 centile lines. Mixed modelling of longitudinal data produced new predictive references for weight centiles of preterm infants. A large population-based prospective study is needed to produce representative longitudinal reference growth charts using these methods

Was the Progenitor of the Sagittarius Stream a Disc Galaxy?

We use N-body simulations to explore the possibility that the Sagittarius (Sgr) dwarf galaxy was originally a late-type, rotating disc galaxy, rather than a non-rotating, pressure-supported dwarf spheroidal galaxy, as previously thought. We find that bifurcations in the leading tail of the Sgr stream, similar to those detected by the SDSS survey, naturally arise in models where the Sgr disc is misaligned with respect to the orbital plane. Moreover, we show that the internal rotation of the progenitor may strongly alter the location of the leading tail projected on the sky, and thus affect the constraints on the shape of the Milky Way dark matter halo that may be derived from modelling the Sgr stream. Our models provide a clear, easily-tested prediction: although tidal mass stripping removes a large fraction of the original angular momentum in the progenitor dwarf galaxy, the remnant core should still rotate with a velocity amplitude ~20 km/s that could be readily detected in future, wide-field kinematic surveys of the Sgr dwarf.Comment: Letter accepted by MNRAS. N-body model animations can be downloaded from http://www.ast.cam.ac.uk/~jorpega/files/sgr

Constructing a man-made c-type cytochrome maquette in vivo:electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo

Joint analysis of X-ray and Sunyaev Zel'dovich observations of galaxy clusters using an analytic model of the intra-cluster medium

We perform a joint analysis of X-ray and Sunyaev Zel'dovich (SZ) effect data using an analytic model that describes the gas properties of galaxy clusters. The joint analysis allows the measurement of the cluster gas mass fraction profile and Hubble constant independent of cosmological parameters. Weak cosmological priors are used to calculate the overdensity radius within which the gas mass fractions are reported. Such an analysis can provide direct constraints on the evolution of the cluster gas mass fraction with redshift. We validate the model and the joint analysis on high signal-to-noise data from the Chandra X-ray Observatory and the Sunyaev-Zel'dovich Array for two clusters, Abell 2631 and Abell 2204.Comment: ApJ in pres

The Scientific Measurement System of the Gravity Recovery and Interior Laboratory (GRAIL) Mission

The Gravity Recovery and Interior Laboratory (GRAIL) mission to the Moon utilized an integrated scientific measurement system comprised of flight, ground, mission, and data system elements in order to meet the end-to-end performance required to achieve its scientific objectives. Modeling and simulation efforts were carried out early in the mission that influenced and optimized the design, implementation, and testing of these elements. Because the two prime scientific observables, range between the two spacecraft and range rates between each spacecraft and ground stations, can be affected by the performance of any element of the mission, we treated every element as part of an extended science instrument, a science system. All simulations and modeling took into account the design and configuration of each element to compute the expected performance and error budgets. In the process, scientific requirements were converted to engineering specifications that became the primary drivers for development and testing. Extensive simulations demonstrated that the scientific objectives could in most cases be met with significant margin. Errors are grouped into dynamic or kinematic sources and the largest source of non-gravitational error comes from spacecraft thermal radiation. With all error models included, the baseline solution shows that estimation of the lunar gravity field is robust against both dynamic and kinematic errors and a nominal field of degree 300 or better could be achieved according to the scaled Kaula rule for the Moon. The core signature is more sensitive to modeling errors and can be recovered with a small margin