Influence of the Valine Zipper Region on the Structure and Aggregation of the Basic Leucine Zipper (bZIP) Domain of Activating Transcription Factor 5 (ATF5)

Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of Activating Transcription Factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and non-reducing conditions. Our data indicate that removal of this region results in a loss of alpha-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation

Aggregation Prediction in Therapeutic Protein Formulations for Excipient Design

Computational Infrastructure & Informatics Poster SessionA major concern in the development therapeutic protein formulations is protein aggregation. Proteins can interact to form bound groups of protein molecules or aggregates. Aggregates in protein formulations reduce effectiveness and can lead to severe immune responses in patients. Excipients are additive molecules that are not therapeutically active, but can increase the stability of protein formulations. An ideal excipient binds with aggregation prone regions on the protein to limit interaction of that region with another protein molecule. The goal of this project is to predict aggregation prone regions and design excipients to interact with these regions. Several tools exist to predict which regions on a protein will be most likely to initiate aggregation. Aggrescan (http://bioinf.uab.es/aggrescan/) and SAP (Spatial Aggregation Potential) were used to predict aggregation prone regions on proteins and the results were compared. Aggrescan uses experimental data to assign each amino acid an aggregation propensity score. An aggregation prone region is identified by a sequence of amino acids with high propensities. The three-dimensional structure is not used in the aggregation prediction. SAP uses molecular simulation to determine regions that are hydrophobic and solvent accessible. Each residue is scored and the results are mapped to the three-dimensional protein structure. A successful prediction tool must use parameters that correlate with aggregation potential for a folded protein. The aggregation prone regions predicted by Aggrescan and SAP were compared to experimental data on protein aggregation. Proteins with a high number of predicted regions or large predicted regions were found to have higher experimental percent aggregation. With the regions identified, molecular simulations were performed for protein-excipient systems. A protein and small molecule docking algorithm was used to determine which regions of the protein certain excipients interacted with. Trehalose, poly(vinylpyrrolidone), and guanadine hydrochloride were used. For an excipient to successfully stabilize a protein and prevent aggregation, the excipient should interact with the aggregation prone regions predicted by Aggrescan and SAP. The predicted regions were compared to the regions where the excipient docks in the molecular simulation. The simulation results were compared to experimental data on the percent aggregation observed in several protein-excipient formulations. The excipients that were found to interact with the predicted aggregation prone regions in simulations should also experimentally prohibit aggregation, leading to lower percent aggregation. Hydrogen-deuterium swapping along with FTIR analysis will be performed experimentally to determine exposed regions on the protein. Proteins with a high number of exposed regions are less stable. The exposed regions will be compared to the aggregation prone regions predicted by Aggrescan and SAP

Spectral Formation in Accreting X-Ray Pulsars: Bimodal Variation of the Cyclotron Energy with Luminosity

Accretion-powered X-ray pulsars exhibit significant variability of the Cyclotron Resonance Scattering Feature (CRSF) centroid energy on pulse-to-pulse timescales, and also on much longer timescales. Two types of spectral variability are observed. For sources in group 1, the CRSF energy is negatively correlated with the variable source luminosity, and for sources in group 2, the opposite behavior is observed. The physical basis for this bimodal behavior is currently not understood. We explore the hypothesis that the accretion dynamics in the group 1 sources is dominated by radiation pressure near the stellar surface, and that Coulomb interactions decelerate the gas to rest in the group 2 sources. We derive a new expression for the critical luminosity such that radiation pressure decelerates the matter to rest in the supercritical sources. The formula for the critical luminosity is evaluated for 5 sources, using the maximum value of the CRSF centroid energy to estimate the surface magnetic field strength. The results confirm that the group 1 sources are supercritical and the group 2 sources are subcritical, although the situation is less clear for those highly variable sources that cross over the critical line. We also explain the variation of the CRSF energy with luminosity as a consequence of the variation of the characteristic emission height. The sign of the height variation is opposite in the supercritical and subcritical cases, hence creating the observed bimodal behavior.Comment: Accepted for publication in Astronomy & Astrophysic

A connection between star formation activity and cosmic rays in the starburst galaxy M 82

Although Galactic cosmic rays (protons and nuclei) are widely believed to be dominantly accelerated by the winds and supernovae of massive stars, definitive evidence of this origin remains elusive nearly a century after their discovery [1]. The active regions of starburst galaxies have exceptionally high rates of star formation, and their large size, more than 50 times the diameter of similar Galactic regions, uniquely enables reliable calorimetric measurements of their potentially high cosmic-ray density [2]. The cosmic rays produced in the formation, life, and death of their massive stars are expected to eventually produce diffuse gamma-ray emission via their interactions with interstellar gas and radiation. M 82, the prototype small starburst galaxy, is predicted to be the brightest starburst galaxy in gamma rays [3, 4]. Here we report the detection of >700 GeV gamma rays from M 82. From these data we determine a cosmic-ray density of 250 eV cm-3 in the starburst core of M 82, or about 500 times the average Galactic density. This result strongly supports that cosmic-ray acceleration is tied to star formation activity, and that supernovae and massive-star winds are the dominant accelerators.Comment: 18 pages, 4 figures; published in Nature; Version is prior to Nature's in-house style editing (differences are minimal

Evidence for long-term Gamma-ray and X-ray variability from the unidentified TeV source HESS J0632+057

HESS J0632+057 is one of only two unidentified very-high-energy gamma-ray sources which appear to be point-like within experimental resolution. It is possibly associated with the massive Be star MWC 148 and has been suggested to resemble known TeV binary systems like LS I +61 303 or LS 5039. HESS J0632+057 was observed by VERITAS for 31 hours in 2006, 2008 and 2009. During these observations, no significant signal in gamma rays with energies above 1 TeV was detected from the direction of HESS J0632+057. A flux upper limit corresponding to 1.1% of the flux of the Crab Nebula has been derived from the VERITAS data. The non-detection by VERITAS excludes with a probability of 99.993% that HESS J0632+057 is a steady gamma-ray emitter. Contemporaneous X-ray observations with Swift XRT reveal a factor of 1.8+-0.4 higher flux in the 1-10 keV range than earlier X-ray observations of HESS J0632+057. The variability in the gamma-ray and X-ray fluxes supports interpretation of the ob ject as a gamma-ray emitting binary.Comment: 8 pages, 3 figures, Accepted for publication in The Astrophysical Journa

VERITAS Observations of the BL Lac Object 1ES 1218+304

The VERITAS collaboration reports the detection of very-high-energy (VHE) gamma-ray emission from the high-frequency-peaked BL Lac object 1ES 1218+304 located at a redshift of z=0.182. A gamma-ray signal was detected with high statistical significance for the observations taken during several months in the 2006-2007 observing season. The photon spectrum between ~160 GeV and ~1.8 TeV is well described by a power law with an index of Gamma = 3.08 +/- 0.34(stat) +/- 0.2(sys). The integral flux above 200 GeV corresponds to ~6% of that of the Crab Nebula. The light curve does not show any evidence for VHE flux variability. Using lower limits on the density of the extragalactic background light (EBL) in the near-IR to mid-IR we are able to limit the range of intrinsic energy spectra for 1ES 1218+304. We show that the intrinsic photon spectrum is harder than a power law with an index of Gamma = 2.32 +/- 0.37. When including constraints from the spectra of 1ES 1101-232 and 1ES 0229+200, the spectrum of 1ES 1218+304 is likely to be harder than Gamma = 1.86 +/- 0.37.Comment: Submitted to Proceedings of "4th Heidelberg International Symposium on High Energy Gamma-Ray Astronomy 2008

VERITAS Observations of the gamma-Ray Binary LS I +61 303

LS I +61 303 is one of only a few high-mass X-ray binaries currently detected at high significance in very high energy gamma-rays. The system was observed over several orbital cycles (between September 2006 and February 2007) with the VERITAS array of imaging air-Cherenkov telescopes. A signal of gamma-rays with energies above 300 GeV is found with a statistical significance of 8.4 standard deviations. The detected flux is measured to be strongly variable; the maximum flux is found during most orbital cycles at apastron. The energy spectrum for the period of maximum emission can be characterized by a power law with a photon index of Gamma=2.40+-0.16_stat+-0.2_sys and a flux above 300 GeV corresponding to 15-20% of the flux from the Crab Nebula.Comment: accepted for publication in The Astrophysical Journa

Status of the VERITAS Observatory

VERITAS, an Imaging Atmospheric Cherenkov Telescope (IACT) system for gammma-ray astronomy in the GeV-TeV range, has recently completed its first season of observations with a full array of four telescopes. A number of astrophysical gamma-ray sources have been detected, both galactic and extragalactic, including sources previously unknown at TeV energies. We describe the status of the array and some highlight results, and assess the technical performance, sensitivity and shower reconstruction capabilities.Comment: Submitted to Proceedings of "4th Heidelberg International Symposium on High Energy Gamma-Ray Astronomy 2008

Detection of Extended VHE Gamma Ray Emission from G106.3+2.7 with VERITAS

We report the detection of very-high-energy (VHE) gamma-ray emission from supernova remnant (SNR) G106.3+2.7. Observations performed in 2008 with the VERITAS atmospheric Cherenkov gamma-ray telescope resolve extended emission overlapping the elongated radio SNR. The 7.3 sigma (pre-trials) detection has a full angular extent of roughly 0.6deg by 0.4deg. Most notably, the centroid of the VHE emission is centered near the peak of the coincident 12CO (J = 1-0) emission, 0.4deg away from the pulsar PSR J2229+6114, situated at the northern end of the SNR. Evidently the current-epoch particles from the pulsar wind nebula are not participating in the gamma-ray production. The VHE energy spectrum measured with VERITAS is well characterized by a power law dN/dE = N_0(E/3 TeV)^{-G} with a differential index of G = 2.29 +/- 0.33stat +/- 0.30sys and a flux of N_0 = (1.15 +/- 0.27stat +/- 0.35sys)x 10^{-13} cm^{-2} s^{-1} TeV^{-1}. The integral flux above 1 TeV corresponds to ~5 percent of the steady Crab Nebula emission above the same energy. We describe the observations and analysis of the object and briefly discuss the implications of the detection in a multiwavelength context.Comment: 5 pages, 2 figure

VERITAS Upper Limit on the VHE Emission from the Radio Galaxy NGC 1275

The recent detection by the Fermi gamma-ray space telescope of high-energy gamma-rays from the radio galaxy NGC 1275 makes the observation of the very high energy (VHE: E > 100 GeV) part of its broadband spectrum particularly interesting, especially for the understanding of active galactic nuclei (AGN) with misaligned multi-structured jets. The radio galaxy NGC 1275 was recently observed by VERITAS at energies above 100 GeV for about 8 hours. No VHE gamma-ray emission was detected by VERITAS from NGC 1275. A 99% confidence level upper limit of 2.1% of the Crab Nebula flux level is obtained at the decorrelation energy of approximately 340 GeV, corresponding to 19% of the power-law extrapolation of the Fermi Large Area Telescope (LAT) result.Comment: Accepted for publication in ApJ Letter