A synchrotron radiation camera and data acquisition system for time resolved X-ray scattering studies

Bordas, J.; Koch, Marta; Clout, Peter; Dorrington, E.; Boulin, C.; Gabriel, A.

doi:10.1088/0022-3735/13/9/011

Cited by 88 publications

(32 citation statements)

References 7 publications

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“…Methods X-ray scattering curves were recorded on the small angle scattering instrument D24 using synchrotron radiation at LURE-DCI (Orsay, France). The instrument (Depautex et al, 1987) and the data-acquisition system (Bordas et al, 1980;Boulin, Kempf, Koch & McLaughlin, 1986) have already been described. The monochromatic beam is focused in the horizontal direction on to the detector in a focal spot typically 1 mm wide using a bent triangular Ge(ll 1) crystal mounted in the compressive Fankuchen geometry with a cut angle c~ = 10.5 ° (Lemonnier, Fourme, Rousseaux & Kahn, 1978).…”

Section: Methodsmentioning

confidence: 99%

Improved Signal-to-Background Ratio in Small-Angle X-ray Scattering Experiments with Synchrotron Radiation using an Evacuated Cell for Solutions

Dubuisson¹,

Décamps²,

Vachette³

1997

J Appl Cryst

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An evacuated, temperature-controlled cell has been built for use on the small-angle X-ray scattering instrument D24 at the synchrotron radiation facility LURE. The sample is placed in a quartz capillary sealed in a stainless-steel holder using a vacuum-tight glue. Several O rings provide a vacuum path upstream and downstream from the cell, so that the X-ray beam only meets the capillary walls and the solution under study between the slits and the beam stop, while the sample is maintained under atmospheric pressure. The cell temperature is controlled via a water circulation through a copper sheath in tight contact with the steel holder. The use of this cell results in a marked reduction of the background, as observed in two series of parallel experiments using a conventional cell and this evacuated cell. The decrease ranges from a factor of 2 at s 1 values larger than 0.008 A,-to more than 15 at s --0.00116A -1, where s is the modulus of the scattering vector (s = 2sin 0/A, 20 is the scattering angle and A is the wavelength of the X-rays).

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Section: Methodsmentioning

confidence: 99%

Improved Signal-to-Background Ratio in Small-Angle X-ray Scattering Experiments with Synchrotron Radiation using an Evacuated Cell for Solutions

Dubuisson¹,

Décamps²,

Vachette³

1997

J Appl Cryst

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“…The instrument (Depautex et al, 1987), the data-acquisition system (Bordas et al, 1980) and the experimental procedures (Fetler et al, 1995) have been described previously. The two-quadrant gas chamber filled with a (90%/10%) mixture of Xe and C2H6 has been built by A. Gabriel and E Dauvergne (EMBL, Grenoble).…”

Section: Solution X-ray Scatteringmentioning

confidence: 99%

Carbamyl Phosphate Modifies the T Quaternary Structure of Aspartate Transcarbamylase, thereby Facilitating the Structural Transition Associated with Cooperativity

Tauc¹,

Vachette²

1997

J Appl Cryst

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The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues, thereby providing a direct means to monitor the amount of different quatemary structures present in solution. Scattering curves in the presence of variable concentrations of several such substrate analogues were recorded using an area detector. Data were analyzed by singular-value decomposition without any prior assumption as to the number of quaternary structure states. They can all be accounted for with only two states. The structural transition appears to be concerted, a conclusion whose validity has been extended to higher resolution and to other combinations of ligands than previously studied using a linear detector. The titration curve with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA) alone is identical to that previously obtained, while that in the additional presence of carbamyl phosphate (CP) shows a clear R shift. However, the scattering pattern of the ATCase-CP complex is shown to be different from the T pattern of the unligated enzyme, though not a linear combination of the Tand R patterns. Therefore, we conclude that CP acts by modifying the quaternary structure of the unligated enzyme to a T-like structure, a structure more easily converted to the R conformation by subsequent addition of PALA. AbbreviationsATCase = aspartate transcarbamylase (or carbamoyltransferase) from E. coli (EC 2.1.3.2.); CP = carbamyl phosphate; PALA = N-(phosphonacetyl)-L-aspartate; T = quaternary structure state in the absence of ligand; R = quaternary structure state in the presence of saturating concentrations of PALA or substrates; SVD = singularvector decomposition; SAXS = small-angle X-ray scattering.

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“…All X-ray scattering curves were recorded on the small angle scattering instrument D24 using synchrotron radiation at LURE-DCI, Orsay, France. The instrument (Depautex et al, 1987), the data acquisition system (Bordas et al, 1980), and the experimental procedures (HervC et al, 1985) have been described previously.…”

Section: Solution X-ray Scatteringmentioning

confidence: 99%

Glu‐50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure

Tauc¹,

Keiser²,

Kantrowitz³

et al. 1994

Protein Science

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Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity highaffinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50 + Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50 + Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steadystate kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50 + Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.Keywords: aspartate carbamoyltransferase; quaternary structure; single amino acid mutation; solution X-ray scattering Aspartate transcarbamoylase (EC 2.1.3.2.) from Escherichia coli catalyzes the first step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and L-aspartate to form Ncarbamoyl-L-aspartate and phosphate. The reaction mechanism is "preferred order" with carbamoyl phosphate binding before aspartate, and N-carbamoylaspartate leaving before phosphate (Hsuanyu & Wedler, 1987). The E. coli enzyme is endowed with

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A synchrotron radiation camera and data acquisition system for time resolved X-ray scattering studies

Cited by 88 publications

References 7 publications

Improved Signal-to-Background Ratio in Small-Angle X-ray Scattering Experiments with Synchrotron Radiation using an Evacuated Cell for Solutions

Improved Signal-to-Background Ratio in Small-Angle X-ray Scattering Experiments with Synchrotron Radiation using an Evacuated Cell for Solutions

Carbamyl Phosphate Modifies the T Quaternary Structure of Aspartate Transcarbamylase, thereby Facilitating the Structural Transition Associated with Cooperativity

Glu‐50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure

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