The Structure of a Complex of Bovine ɑ-Thrombin and Recombinant Hirudin at 2.8-Å Resolution

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme

The Structure of a Complex of Bovine &-Thrombin and Recombinant Hirudin at 2.8-A Resolution

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme

The Structure of a Complex of Bovine &-Thrombin and Recombinant Hirudin at 2.8-A Resolution

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme

CODE-2 : moored array and large-scale data report

The Coastal Ocean Dynamics Experiment (CODE) was undertaken to identify and study the important dynamical processes which govern the wind-driven motion of coastal water over the continental shelf. The initial effort in this multi-year, multi-institutional research program was to obtain high-quality data sets of all the relevant physical variables needed to construct accurate kinematic and dynamic descriptions of the response of shelf water to strong wind forcing in the 2 to 10 day band. A series of two small-scale, densely- instrumented field experiments of approximately four months duration (called CODE-1 and CODE-2) were designed to explore and to determine the kinematics and momentum and heat balances of the local wind-driven flow over a region of the northern California shelf which is characterized by both relatively simple bottom topography and large wind stress events in both winter and summer. A more lightly instrumented, long -term, large-scale component was designed to help separate the local wind-driven response in the region of the small-scale experiments from motions generated either offshore by the California Current system or in some distant region along the coast, and also to help determine the seasonal cycles of the atmospheric forcing, water structure, and coastal currents over the northern California shelf. The first small-scale experiment (CODE-1) was conducted between April and August, 1981 as a pilot study in "which primary emphasis was placed on characterizing the wind-driven "signal" and the "noise" from which this signal must be extracted. In particular, CODE-1 was designed to identify the key features of the circulation and its variability over the northern California shelf and to determine the important time and length scales of the wind-driven response. The second small-scale experiment (CODE-2) was conducted between April and August, 1982 and was designed to sample more carefully the mesoscale horizonta1 variability observed in CODE-1. This report presents a basic description of the moored array data and some other Eulerian data collected during CODE-2. A brief description of the CODE-2 field program is presented first, followed by a description of the common data analysis procedures used to produce the various data sets presented here. Then basic descriptions of the following data sets are presented: (a) the coastal and moored meteorological measurements, (b) the moored current measurements, (c) array plots of the surface wind stress and near-surface current measurements, (d) the moored temperature and conductivity observations, (e) the bottom pressure measurements, and (f) the wind and adjusted coastal sea level observations obtained as part of the CODE-2 large-scale component.This work has been supported by the National Science Foundation

Host and Viral Proteins in the Virion of Kaposi's Sarcoma-Associated Herpesvirus

Infection of cultured cells with Kaposi's sarcoma associated herpesvirus (KSHV) typically establishes a latent infection, in which only a few viral genes are expressed. Recently, it has been reported that a subset of lytic genes are transiently expressed very early after viral entry but that this burst of abortive lytic gene expression is terminated with the supervention of latency (H. H. Krishnan, P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, and B. Chandran, J. Virol. 78:3601-3620, 2004). To identify molecules imported into cells by KSHV that might influence this gene expression program, we have examined the protein composition of the KSHV particle. Immunoblotting of virus particles demonstrated that RTA, the lytic switch protein, and RAP, a viral protein that is a transcriptional and cell cycle modulator, were both incorporated into virus particles. In a second approach, polypeptides isolated from purified virions were identified by mass-spectrometric analysis of their constituent tryptic peptides. With this approach we were able to identify 18 major virion proteins, including structural, regulatory, and signaling proteins of both viral and cellular origin

Gap flow in an Alpine valley during a shallow south föhn event: Observations, numerical simulations and hydraulic analogue

International audienceThis paper examines the three-dimensional structure and dynamics of southerly hybrid gap/mountain ow through the Wipp valley (Wipptal), Austria, observed on 30 October 1999 using high-resolution observations and model simulations. The observations were obtained during a shallow south föhn event documented in the framework of the Mesoscale Alpine Programme (MAP). Three important data sources were used: the airborne differential-absorption lidar LEANDRE 2, the ground-based Doppler lidar TEACO2 and in situ measurements from the National Oceanic and Atmospheric Administration P-3 aircraft. This event was simulated down to 2 km horizontal resolution using the non-hydrostatic mesoscale model Meso-NH. The structure and dynamics of the ow were realistically simulated. The combination of high-resolution observations and numerical simulations provided a comprehensive three-dimensional picture of the ow through the Wipptal: in the gap entrance region (Brenner Pass, Austria), the low-level jet was not solely due to the channelling of the southerly synoptic ow through the elevated gap. Part of the Wipptal ow originated as a mountain wave at the valley head wall of the Brenner Pass. Downstream of the pass, the shallow föhn ow had the characteristics of a downslope windstorm as it rushed down towards the Inn valley (Inntal) and the City of Innsbruck, Austria. Downhill of the Brenner Pass, the strongest ow was observed over a small obstacle along the western side wall (the Nösslachjoch), rather than channelled in the deeper part of the valley just to the east. Further north, the low-level jet was observed in the centre of the valley. Approximately halfway between Brenner Pass and Innsbruck, where the along-axis direction of the valley changes from north to north-northwest , the low-level jet was observed to be de ected to the eastern side wall of the Wipptal. Interaction between the Stubaier Alpen (the largest and highest topographic feature to the west of the Wipptal) and the south-westerly synoptic ow was found to be the primary mechanism responsible for the dee ection. The along-and cross-valley structure and dynamics of the ow were observed to be highly variable due to the in uence of surrounding mountains, localized steep slopes within the valley and outt ows from tributaries (the Gschnitztal and the Stubaital) to the west of the Wipptal. For that shallow föhn case, observations and simulations provided a large body of evidence that downslope ow created thinning/ thickening uid and accelerations/ decelerations reminiscent of mountain wave/hydraulic theory. Along the Wipptal, two hydraulic-jump-like transitions were observed and simulated, (i) on the lee slope of the Nösslachjoch and (ii) in the Gschnitztal exit region. A hydraulic solution of the ow was calculated in the framework of reduced-gravity shallow-water theory. The down-valley evolution of the Froude number computed using LEANDRE 2, P-3 ight level and TEACO2 measurements conn rmed that these transitions were associated with super-to subcritical transitions