Modeling Disordered Regions in Proteins Using Rosetta

Protein structure prediction methods such as Rosetta search for the lowest energy conformation of the polypeptide chain. However, the experimentally observed native state is at a minimum of the free energy, rather than the energy. The neglect of the missing configurational entropy contribution to the free energy can be partially justified by the assumption that the entropies of alternative folded states, while very much less than unfolded states, are not too different from one another, and hence can be to a first approximation neglected when searching for the lowest free energy state. The shortcomings of current structure prediction methods may be due in part to the breakdown of this assumption. Particularly problematic are proteins with significant disordered regions which do not populate single low energy conformations even in the native state. We describe two approaches within the Rosetta structure modeling methodology for treating such regions. The first does not require advance knowledge of the regions likely to be disordered; instead these are identified by minimizing a simple free energy function used previously to model protein folding landscapes and transition states. In this model, residues can be either completely ordered or completely disordered; they are considered disordered if the gain in entropy outweighs the loss of favorable energetic interactions with the rest of the protein chain. The second approach requires identification in advance of the disordered regions either from sequence alone using for example the DISOPRED server or from experimental data such as NMR chemical shifts. During Rosetta structure prediction calculations the disordered regions make only unfavorable repulsive contributions to the total energy. We find that the second approach has greater practical utility and illustrate this with examples from de novo structure prediction, NMR structure calculation, and comparative modeling

OH and OI airglow layer modulation by ducted shortperiod gravity waves: effects of trapping altitude

Perturbations to the OH and OI [O(1S) 557.7 nm] airglow layers by ducted gravity waves near the Brunt-Väisälä period are investigated using a 2-D numerical model. Airglow signatures of these waves are strongly determined by perturbations of O, O3, and H, which exhibit peak densities near and above mesopause. Strong periodic vertical wind components of short-period gravity waves induce opposite relative density perturbations above and below the layer density peaks. Airglow signatures for ducted waves depend on the specific vertical shapes and altitudes of the wave packets relative to ambient species density profiles; waves perturbing only the bottoms or tops of the layers produce signatures differing from those able to perturb the entire layer thickness. Line-of-sight cancellation occurs between opposite perturbations above and below airglow layer peaks, even for standing waves without vertical phase progression. Integrated brightness-weighted temperature and intensity can thus appear in-phase or antiphase for standing waves, depending on the wave-packet altitude relative to the density gradients. Comparisons of OH and OI layer intensities also reveal in-phase or antiphase relative intensity responses and do not directly indicate the phase of the wave perturbations at layer peak altitudes. Despite this ambiguity, simultaneous brightness-weighted temperature measurements may provide additional insight into wave structure, amplitude, and trapping altitude. For waves of sufficient amplitude that perturb steep density gradients, nonlinearity of the airglow response may be observable; this effect is most prominent when strong cancellation of the linear signature occurs

An atmospheric radiation model for Cerro Paranal. I. The optical spectral range

The Earth's atmosphere affects ground-based astronomical observations. Scattering, absorption, and radiation processes deteriorate the signal-to-noise ratio of the data received. For scheduling astronomical observations it is, therefore, important to accurately estimate the wavelength-dependent effect of the Earth's atmosphere on the observed flux. In order to increase the accuracy of the exposure time calculator of the European Southern Observatory's (ESO) Very Large Telescope (VLT) at Cerro Paranal, an atmospheric model was developed as part of the Austrian ESO In-Kind contribution. It includes all relevant components, such as scattered moonlight, scattered starlight, zodiacal light, atmospheric thermal radiation and absorption, and non-thermal airglow emission. This paper focuses on atmospheric scattering processes that mostly affect the blue (< 0.55 mum) wavelength regime, and airglow emission lines and continuum that dominate the red (> 0.55 mum) wavelength regime. While the former is mainly investigated by means of radiative transfer models, the intensity and variability of the latter is studied with a sample of 1186 VLT FORS1 spectra. For a set of parameters such as the object altitude angle, Moon-object angular distance, ecliptic latitude, bimonthly period, and solar radio flux, our model predicts atmospheric radiation and transmission at a requested resolution. A comparison of our model with the FORS1 spectra and photometric data for the night-sky brightness from the literature, suggest a model accuracy of about 20%. This is a significant improvement with respect to existing predictive atmospheric models for astronomical exposure time calculators.Comment: 23 pages, 21 figures, accepted for publication in A&

Unexpected Occurrence of Mesospheric Frontal Gravity Wave Events Over South Pole (90°S)

Since 2010, Utah State University has operated an infrared Advanced Mesospheric Temperature Mapper at the Amundsen–Scott South Pole station to investigate the upper atmosphere dynamics and temperature deep within the vortex. A surprising number of “frontal” gravity wave events (86) were recorded in the mesospheric OH(3,1) band intensity and rotational temperature images (typical altitude of ~87 km) during four austral winters (2012–2015). These events are gravity waves (GWs) characterized by a sharp leading wave front followed by a quasi-monochromatic wave train that grows with time. A particular subset of frontal gravity wave events has been identified in the past (Dewan & Picard, 1998) as “bores.” These are usually associated with wave ducting within stable mesospheric inversion layers, which allow them to propagate over very large distances. They have been observed on numerous occasions from low-latitude and midlatitude sites, but to date, very few have been reported at high latitudes. This study provides new analyses of the characteristics of frontal events at high latitudes and shows that most of them are likely ducted. The occurrence of these frontal GW events over this isolated region strongly supports the existence of horizontally extensive mesospheric thermal inversion layers over Antarctica, leading to regions of enhanced stability necessary for GW trapping and ducting

Histone H3 Localizes to the Centromeric DNA in Budding Yeast

During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.National Institutes of Health grant: (GM 074150); Ludwig Institute for Cancer Research; European Molecular Biology Organization (EMBO) long-term fellowship

Stressful situation if CENP-A not front and CENter

The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1$^{CENP-U}$ and Okp1$^{CENP-Q}$ form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers