Structural Analysis of Diheme Cytochrome c by Hydrogen–Deuterium Exchange Mass Spectrometry and Homology Modeling
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Ying Zhang↗
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Mechanosensitive Ion Channels in Bacteria: Functional Domains and Mechanisms of Gating
The past funding period was productive for the group. The progress in the mechanosensitive channel field was critically affected in the end of 1998 by the solution of the crystal structure of the mycobacterial homolog of MscL by our colleagues from Caltech. Having the structure of TbMscL in the closed state, we developed a detailed homology model of EcoMscL, and related the structural model with the wealth of functional phenomenology available for the E. coli version of the channel (EcoMscL). The biophysical properties of the open MscL helped to model the open conformation and infer the pathway for the entire gating transition. The following experiments provided strong support to the atomic model of the gating process, and allowed to make further predictions. The work has advanced our understanding of tension-driven conformational transitions in membrane-embedded mechanosensory proteins, determine major energetic contributions and set the stage for further exploration of the whole family of mechanosensitive channels. The results have been published in seven experimental and theoretical papers, with three other papers currently in press or in preparation.
Sukharev, Sergei↗
Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase
The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.
Non-NASA Center↗
Modeling Non-homologous End Joining
Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several NHEJ proteins such as Ku, DNA-PKcs, XRCC4, Ligase IV and so on. Once DSBs are generated, Ku is first recruited to the DNA end, followed by other NHEJ proteins for DNA end processing and ligation. Because of the direct ligation of break ends without the need for a homologous template, NHEJ turns out to be an error-prone but efficient repair pathway. Some mechanisms have been proposed of how the efficiency of NHEJ repair is affected. The type of DNA damage is an important factor of NHEJ repair. For instance, the length of DNA fragment may determine the recruitment efficiency of NHEJ protein such as Ku [1], or the complexity of the DNA breaks [2] is accounted for the choice of NHEJ proteins and subpathway of NHEJ repair. On the other hand, the chromatin structure also plays a role of the accessibility of NHEJ protein to the DNA damage site. In this talk, some mathematical models of NHEJ, that consist of series of biochemical reactions complying with the laws of chemical reaction (e.g. mass action, etc.), will be introduced. By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed
Li, Yongfeng↗
Three-Dimensional Modeling of Quasi-Homologous Solar Jets
Recent solar observations (e.g., obtained with Hinode and STEREO) have revealed that coronal jets are a more frequent phenomenon than previously believed. This higher frequency results, in part, from the fact that jets exhibit a homologous behavior: successive jets recur at the same location with similar morphological features. We present the results of three-dimensional (31)) numerical simulations of our model for coronal jets. This study demonstrates the ability of the model to generate recurrent 3D untwisting quasi-homologous jets when a stress is constantly applied at the photospheric boundary. The homology results from the property of the 3D null-point system to relax to a state topologically similar to its initial configuration. In addition, we find two distinct regimes of reconnection in the simulations: an impulsive 3D mode involving a helical rotating current sheet that generates the jet, and a quasi-steady mode that occurs in a 2D-like current sheet located along the fan between the sheared spines. We argue that these different regimes can explain the observed link between jets and plumes.
Pariat, E.↗
Expanded Very Large Array Nova Project Observations of the Classical NovaV1723 Aquilae
We present radio light curves and spectra of the classical nova VI723 Aql obtained with the Expanded Very Large Array (EVLA). This is the first paper to showcase results from the EVLA Nova Project, which comprises a team of observers and theorists utilizing the greatly enhanced sensitivity and frequency coverage of EVLA radio observations, along with observations at other wavelengths, to reach a deeper understanding of the energetics, morphology, and temporal characteristics of nova explosions. Our observations of VI723 Aql span 1-37 GHz in frequency, and we report on data from 14 to 175 days following the time of the nova explosion. The broad frequency coverage and frequent monitoring show that the radio behavior of VI723 Aql does not follow the classic Hubble-flow model of homologous spherically expanding thermal ejecta. The spectra are always at least partially optically thin, and the flux rises on faster timescales than can be reproduced with linear expansion. Therefore, any description of the underlying physical processes must go beyond this simple picture. The unusual spectral properties and light curve evolution might be explained by multiple emitting regions or shocked material. Indeed, X-ray observations from Swift reveal that shocks are likely present.
Krauss, Miriam I.↗
A comparative modeling of supernova 1993J
The light curve of Supernova 1993J is calculated using two computational radiation transport approaches. The two approaches are represented by the computer codes STELLA and EDDINGTON. The emphasis is on the shock breakout and the photometry in the U, B and V bands during the first 120 days of the supernova. The STELLA model includes implicit hydrodynamics and is able to model early supernova evolution before the expansion is homologous. The STELLA model employs multi-group photonics and is able to follow the radiation as it decouples from the matter. The EDDINGTON code uses an algorithm for integrating the transport equation which assumes homologous expansion and uses a finer frequency resolution. The agreement between the two codes is considered to be satisfactory only in the case where compatible physical assumptions are made concerning the opacity. The assumptions are justified. The continuum spectrum for SN 1993J is predicted near the shock breakout to be superior to that predicted by standard single energy group hydrocodes. The uncertainties involved in current time dependent models of supernova light curves are discussed.
Blinnikov, Sergei↗
Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining
We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.
Cucinotta, Francis, A.↗
Intricate Crystal Structure of Dihydrolipoamide Dehydrogenase (E3) with its Binding Protein: Multiple Copies, Dynamic and Static Disorders
Human E3 and binding protein E3BP are two components of the pyruvate dehydrogenase complex. Crystallization of E3 with 221-amino acid fragment of E3BP (E3BPdd) led to crystals that diffracted to a resolution of 2.6 Angstroms. Structure determination involved molecular replacement using a dimer of E3 homolog as a search model and de novo building of the E3BPdd peptide. Solution was achieved by inclusion of one E3 dimer at a time, followed by refinement until five E3 dimers were located. This complete content of E3 provided electron density maps suitable for tracing nine peptide chains of E3BPdd, eight of them being identified with partial occupancies. Final content of the asymmetric unit consists of five E3 dimers, each binding one E3BPdd molecule. In four of these molecular complexes, E3BPdd is in static disorder resulting in E3BPdd binding to either one or the other monomer of the E3 dimer. However, E3BPdd of the fifth E3 dimer forms specific contacts that lock it at one monomer. In addition to this static disorder, E3BPdd reveals high mobility in the limited space of the crystal lattice. Support from NIH and NASA.
Makal, A.↗
The structure of horizontal-branch models. I - The zero-age horizontal branch
A detailed study of the structure of zero-age horizontal-branch (ZAHB) models is studied in order to show how the hydrostatic structure of these models changes with the input parameters and determines the H-R diagram location of a given model. The properties of composite polytropes on the homology-invariant (U,V)-plane are demonstrated. A variety of test models and sequences were constructed to elucidate the underlying factors that give rise to the wide variation in HB model properties with composition. The roles of the CNO elements as nuclear catalysts and of the envelope sources, as well as the envelope helium abundance are reexamined. It is found that, for stars of a fixed range of mass arriving on the HB, the stellar distribution is determined mainly by CNO for low metallicities (Fe/H of less than about -1), but mainly by opacity sources for high metallicities. The value of Fe/H where CNO ceases to dominate depends significantly on the adopted opacity and will decrease if and when opacity estimates are revised upward.
Dorman, Ben↗
EIT Crinkles as Evidence for the Breakout Model of Solar Eruptions
We present observations of two homologous flares in NOAA Active Region 8210 occurring on 1998 May 1 and 2, using EUV data from the EUV Imaging Telescope (EIT) on board the Solar and Heliospheric Observatory, high-resolution and high-time cadence images from the soft X-ray telescope on Yohkoh, images or fluxes from the hard X-ray telescope on Yohkoh and the BATSE experiment on board the Compton Gamma Ray Observatory, and Ca(XIX) soft X-ray spectra from the Bragg crystal spectrometer (BCS) on Yohkoh. Magnetograms indicate that the flares occurred in a complex magnetic topology, consisting of an emerging flux region (EFR) sandwiched between a sunspot to the west and a coronal hole to the east. In an earlier study we found that in EIT images, both flaring episodes showed the formation of a crinkle-like pattern of emission (EIT crinkles) occurring in the coronal hole vicinity, well away from a central 'core field' area near the EFR-sunspot boundary. With our expanded data set, here we find that most of the energetic activity occurs in the core region in both events, with some portions of the core brightening shortly after the onset of the EIT crinkles, and other regions of the core brightening several minutes later, coincident with a burst of hard X-rays; there are no obvious core brightenings prior to the onset of the EIT crinkles. These timings are consistent with the 'breakout model' of solar eruptions, whereby the emerging flux is initially constrained by a system of overlying magnetic field lines, and is able to erupt only after an opening develops in the overlying fields as a consequence of magnetic reconnection at a magnetic null point. In our case, the EIT crinkles would be a signature of this pre-impulsive phase magnetic reconnection, and brightening of the core only occurs after the core fields begin to escape through the newly created opening in the overlying fields. Morphology in soft X-ray images and properties in hard X-rays differ between the two events, with complexities that preclude a simple determination of the dynamics in the core at the times of eruption. From the BCS spectra, we find that the core region expends energy at a rate of approximately 10(exp 26) ergs/s during the time of the growth of the EIT crinkles; this rate is an upper limit to energy expended in the reconnections opening the overlying fields. Energy losses occur at an order of magnitude higher rate near the time of the peak of the events. There is little evidence of asymmetry in the spectra, consistent with the majority of the mass flows occurring normal to the line of sight. Both events have similar electron temperature dependencies on time.
Sterling, Alphonse C.↗
EIT Crinkles as Evidence for the Breakout Model of Solar Eruptions
We present observations of two homologous flares in NOAA active region 8210 occurring on 1998 May 1 and May 2, using EUV data from the Extreme Ultraviolet Radiation Imaging Telescope (EIT) on the Solar and Heliospheric Observatory (SOHO), high-resolution and high-time cadence images from the soft X-ray telescope (SXT) on Yohkoh, images or fluxes from the hard X-ray telescope (HXT) on Yohkoh and the BATSE experiment on the Compton Gamma Ray Observatory (CGRO), and Ca xix soft X-ray spectra from the Bragg crystal spectrometer (BCS) on Yohkoh. Magnetograms indicate that the flares occurred in a complex magnetic topology, consisting of an emerging flux region (EFR) sandwiched between a sunspot to the west and a coronal hole to the east. In an earlier study we found that in EIT images, both flaring episodes showed the formation of a crinkle-like pattern of emission ("EIT crinkles") occurring in the coronal hole vicinity, well away from a central "core field" area near the EFR-sunspot boundary. With our expanded data set, here we find that most of the energetic activity occurs in the core region in both events, with some portions of the core brightening shortly after the onset of the EIT crinkles, and other regions of the core brightening several minutes later, coincident with a burst of hard X-rays: there are no obvious core brightenings prior to the onset of the EIT crinkles. These timings are consistent with the "breakout model" of solar eruptions, whereby the emerging flux is initially constrained by a system of overlying magnetic field lines, and is able to erupt only after an opening develops in the overlying fields as a consequence of magnetic reconnection at a magnetic null point. In our case, the EIT crinkles would be a signature of this pre-impulsive-phase magnetic reconnection, and brightening of the core only occurs after the core fields begin to escape through the newly-created opening in the overlying fields. Morphology in soft X-ray images and properties in hard X-rays differ between the two events, with complexities that preclude a simple determination of the dynamics in the core at the times of eruption. From the BCS spectra, we find that the core region expends energy at a rate of approx. 10(exp 26) erg per second during the time of the growth of the EIT crinkles; this rate is an upper limit to energy expended in the reconnections opening the overlying fields. Energy losses occur at an order-of-magnitude higher rate near the time of the peak of the events. There is little evidence of asymmetry in the spectra, consistent with the majority of the mass flows occurring normal to the line-of-sight. Both events have similar electron temperature dependencies on time.
Sterling, Alphonse C.↗
Non-LTE model atmosphere analysis of Nova Cygni 1992
We use spherically symmetric non-local thermodynamic equilibrium (non-LTE), line-blanketed, expanding model atmospheres to analyze the International Ultraviolet Explorer (IUE) and optical spectra of Nova Cygni 1992 during the early phases of its outburst. We find that the first IUE spectrum obtained just after discovery on 1992 February 20, is best reproduced by a model atmosphere with a steep density gradient and homologous expansion, whereas the IUE and optical spectra obtained on February 24 show an extended, optically thick, wind structure. Therefore, we distinguish two phases of the early evolution of the nova photosphere: the initial, rapid, 'fireball' phase and the subsequent, much longer, optically thick 'wind' phase. The importance of line-blanketing in nova spectra is demonstrated. Our preliminary abundance analysis implies that hydrogen is depeleted in the ejecta, corresponding to abundance enhancements of Fe by a factor of approximately 2 and of CNO by more than a factor of 10 when compared to solar abundances. The synthetic spectra reproduce both the observed pseudo-continua as well as most of the observed features from the UV to the optical spectral range and demonstrate the importance of obtaining nearly simultaneous UV and optical spectra for performing accurate analyses of expanding stellar atmospheres (for both novae and supernovae).
Hauschildt, P. H.↗
Signaling in Human and Murine Lymphocytes in Microgravity: Parallels and Contrasts
Immune function in space undergoes dramatic changes, some of which are detrimental to lymphocyte function. These changes may lead to significant immune suppression. Studies with human lymphocytes both in space flight and with ground-based models (NASA in vitro ground-based microgravity analog) indicate that T cell activation is inhibited in microgravity. Other lymphocyte functions, such as locomotion, are also inhibited. There is about an 80 percent homology in the immune response of mice to that of humans. A murine model was investigated because of its ability to parallel some microgravity using hind limb suspension. In in vivo antiorthostatically (AOS)-suspended mice, T cell activation is greatly suppressed, with the majority of activation related cytokines being inhibited. PHA activation in lymphocytes derived from AOS mice (in vivo ground-based microgravity analog) is also suppressed. Calcium ionophore studies in human lymphocytes exposed to modeled microgravity indicate that the calcium pathways are probably unaffected in microgravity. IP3 (inositol triphosphate) receptor expression in both human and mouse lymphocytes cultured in modeled microgravity indicate no suppression of calcium signaling. In the human system, microgravity seems to inhibit signaling cascades either at the level of, or up-stream of, Protein Kinase C (PKC). In particular, a membrane event, such as phospholipase C gamma 1 activity in human lymphocytes is affected, with its direct upstream effector, LAT, being deficiently expressed. In the mouse pathway, LAT is undiminished while another critical intermediate, SLP-76, is diminished significantly. This study identifies critical stages in the human and mouse immune systems and in lymphocytes as a function of microgravity.
Neal, Pellis↗
Developmental genes and the origin and evolution of Metazoa
The author discusses the role of developmental genes in evolution of life and the use of molecular approaches to confirm morphologic homology, to aid in understanding developmental mechanisms of evolution of novel structures, and to investigate some aspects of the evolution of life. Topics examined include developmental genes and homology; the role of co-optation and divergence, homeosis, and heterochrony as mechanisms of evolution; and life history evolution and model organisms.
NASA Discipline Exobiology↗
Multistep modeling of protein structure: application to bungarotoxin
Modelling of bungarotoxin in atomic details is presented in this article. The model-building procedure utilizes the low-resolution crystal coordinates of the c-alpha atoms of bungarotoxin, sequence homology within the neurotoxin family, as well as high-resolution x-ray diffraction data of cobratoxin and erabutoxin. Our model-building procedure involves: (a) principles of comparative modelling, (b) embedding procedures of distance geometry, and (c) use of molecular mechanics for optimizing packing. The model is not only consistent with the c-alpha coordinates of crystal structure, but also agrees with solution conformational features of the triple-stranded beta sheet as observed by NOE measurements.
NASA Discipline Exobiology↗
Worst case estimation of homology design by convex analysis
The methodology of homology design is investigated for optimum design of advanced structures. for which the achievement of delicate tasks by the aid of active control system is demanded. The proposed formulation of homology design, based on the finite element sensitivity analysis, necessarily requires the specification of external loadings. The formulation to evaluate the worst case for homology design caused by uncertain fluctuation of loadings is presented by means of the convex model of uncertainty, in which uncertainty variables are assigned to discretized nodal forces and are confined within a conceivable convex hull given as a hyperellipse. The worst case of the distortion from objective homologous deformation is estimated by the Lagrange multiplier method searching the point to maximize the error index on the boundary of the convex hull. The validity of the proposed method is demonstrated in a numerical example using the eleven-bar truss structure.
Yoshikawa, N.↗
Scaling effects in the impact response of graphite-epoxy composite beams
In support of crashworthiness studies on composite airframes and substructure, an experimental and analytical study was conducted to characterize size effects in the large deflection response of scale model graphite-epoxy beams subjected to impact. Scale model beams of 1/2, 2/3, 3/4, 5/6, and full scale were constructed of four different laminate stacking sequences including unidirectional, angle ply, cross ply, and quasi-isotropic. The beam specimens were subjected to eccentric axial impact loads which were scaled to provide homologous beam responses. Comparisons of the load and strain time histories between the scale model beams and the prototype should verify the scale law and demonstrate the use of scale model testing for determining impact behavior of composite structures. The nonlinear structural analysis finite element program DYCAST (DYnamic Crash Analysis of STructures) was used to model the beam response. DYCAST analysis predictions of beam strain response are compared to experimental data and the results are presented.
Jackson, Karen E.↗