Designing protein β-sheet surfaces by Z-score optimization

Studies of lattice models of proteins have suggested that the appropriate energy expression for protein design may include nonthermodynamic terms to accommodate negative design concerns. One method, developed in lattice model studies, maximizes a quantity known as the "Z-score," which compares the lowest energy sequence whose ground state structure is the target structure to an ensemble of random sequences. Here we show that, in certain circumstances, the technique can be applied to real proteins. The resulting energy expression is used to design the β-sheet surfaces of two real proteins. We find experimentally that the designed proteins are stable and well folded, and in one case is even more thermostable than the wild type

An improved map of conserved regulatory sites for Saccharomyces cerevisiae

BACKGROUND: The regulatory map of a genome consists of the binding sites for proteins that determine the transcription of nearby genes. An initial regulatory map for S. cerevisiae was recently published using six motif discovery programs to analyze genome-wide chromatin immunoprecipitation data for 203 transcription factors. The programs were used to identify sequence motifs that were likely to correspond to the DNA-binding specificity of the immunoprecipitated proteins. We report improved versions of two conservation-based motif discovery algorithms, PhyloCon and Converge. Using these programs, we create a refined regulatory map for S. cerevisiae by reanalyzing the same chromatin immunoprecipitation data. RESULTS: Applying the same conservative criteria that were applied in the original study, we find that PhyloCon and Converge each separately discover more known specificities than the combination of all six programs in the previous study. Combining the results of PhyloCon and Converge, we discover significant sequence motifs for 36 transcription factors that were previously missed. The new set of motifs identifies 636 more regulatory interactions than the previous one. The new network contains 28% more regulatory interactions among transcription factors, evidence of greater cross-talk between regulators. CONCLUSION: Combining two complementary computational strategies for conservation-based motif discovery improves the ability to identify the specificity of transcriptional regulators from genome-wide chromatin immunoprecipitation data. The increased sensitivity of these methods significantly expands the map of yeast regulatory sites without the need to alter any of the thresholds for statistical significance. The new map of regulatory sites reveals a more elaborate and complex view of the yeast genetic regulatory network than was observed previously

Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development

Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that this is required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional type IV pili are required mechanosensory elements. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well

Registry in a tube:multiplexed pools of retrievable parts for genetic design space exploration

The publisher would like to apologise for an error in Figure 4. The shaded gates in Figure 4C and 4D were missing in the final version. These shaded gates illustrate that any combination of repressors can be wired together by accessing the pools in Figure 4A. The correct figures are available below and have been replaced in the published article

Asymmetry and Inequity in the Inheritance of a Bacterial Adhesive

Pseudomonas aeruginosa is an opportunistic human pathogen that forms biofilm infections in a wide variety of contexts. Biofilms initiate when bacteria attach to a surface, which triggers changes in gene expression leading to the biofilm phenotype.Wehave previously shown, for the P. aeruginosa lab strain PAO1, that the self-produced polymer Psl is the most dominant adhesive for attachment to the surface but that another self-produced polymer, Pel, controls the geometry of attachment of these rod-shaped bacteria—strains that make Psl but not Pel are permanently attached to the surface but adhere at only one end (tilting up off the surface), whereas wild-type bacteria that make both Psl and Pel are permanently attached and lie down flat with very little or no tilting (Cooley et al 2013 Soft Matter 9 3871–6). Here we show that the change in attachment geometry reflects a change in the distribution of Psl on the bacterial cell surface. Bacteria that make Psl and Pel have Psl evenly coating the surface, whereas bacteria that make only Psl have Psl concentrated at only one end.Weshow that Psl can act as an inheritable, epigenetic factor. Rod-shaped P. aeruginosa grows lengthwise and divides across the middle.Wefind that asymmetry in the distribution of Psl on a parent cell is reflected in asymmetry between siblings in their attachment to the surface. Thus, Pel not only promotes P. aeruginosa lying downWe thank Professor Matthew Parsek (University of Washington, Seattle) for his generous gift of bacterial PAO1 strains.Wealso thank Professor Marvin Whiteley (University of Texas at Austin) forWTandΔpsl polysaccharide preparations. SIM imaging (for figure 1) was performed in the Microscopy Core Facility within the Institute for Cellular and Molecular Biology atUTAustin, with the assistance of Julie Hayes. This work was funded by startup funds fromUTAustin and a gift from ExxonMobil to VDG, and by a grant from the Human Frontiers Science Program (HFSP RGY0081/2012-GORDON).Center for Nonlinear Dynamic

The Panchromatic Hubble Andromeda Treasury. Progression of Large-Scale Star Formation across Space and Time in M31

We investigate the clustering of early-type stars younger than 300 Myr on galactic scales in M31. Based on the stellar photometric catalogs of the Panchromatic Hubble Andromeda Treasury program that also provides stellar parameters derived from the individual energy distributions, our analysis is focused on the young stars in three star-forming regions, located at galactocentric distances of about 5, 10, and 15 kpc, corresponding to the inner spiral arms, the ring structure, and the outer arm, respectively. We apply the two-point correlation function to our selected sample to investigate the clustering behavior of these stars across different time- and length-scales. We find that young stellar structure survives across the whole extent of M31 longer than 300 Myr. Stellar distribution in all regions appears to be self-similar, with younger stars being systematically more strongly clustered than the older, which are more dispersed. The observed clustering is interpreted as being induced by turbulence, the driving source for which is probably gravitational instabilities driven by the spiral arms, which are stronger closer to the galactic centre.Comment: 10 pages, 5 figures. To appear in "LESSONS FROM THE LOCAL GROUP - A Conference in Honour of David Block and Bruce Elmegreen" eds. Freeman, K.C., Elmegreen, B.G., Block, D.L. & Woolway, M. (Springer: New York

p53-Dependent Transcriptional Responses to Interleukin-3 Signaling

p53 is critical in the normal response to a variety of cellular stresses including DNA damage and loss of p53 function is a common feature of many cancers. In hematological malignancies, p53 deletion is less common than in solid malignancies but is associated with poor prognosis and resistance to chemotherapy. Compared to their wild-type (WT) counterparts, hematopoietic progenitor cells lacking p53 have a greater propensity to survive cytokine loss, in part, due to the failure to transcribe Puma, a proapoptotic Bcl-2 family member. Using expression arrays, we have further characterized the differences that distinguish p53−/− cells from WT myeloid cells in the presence of Interleukin-3 (IL-3) to determine if such differences contribute to the increased clonogenicity and survival responses observed in p53−/− cells. We show that p53−/− cells have a deregulated intracellular signaling environment and display a more rapid and sustained response to IL-3. This was accompanied by an increase in active ERK1/2 and a dependence on an intact MAP kinase signaling pathway. Contrastingly, we find that p53−/− cells are independent on AKT for their survival. Thus, loss of p53 in myeloid cells results in an altered transcriptional and kinase signaling environment that favors enhanced cytokine signaling