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Acta Crystallographica Section F: Structural Biology and Crystallization Communications (1)
Journal of Experimental Botany (1)
Pflug, Alexander (2)
Brown, Naomi J. (1)
Engh, Richard A. (1)
Gowik, Udo (1)
Hibberd, Julian M. (1)
Johnson, Kenneth A. (1)
Liu, Zheng (1)
Newell, Christine A. (1)
Westhoff, Peter (1)
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Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89
Johnson, Kenneth A.
Engh, Richard A.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications
The anomalous dispersion signal of the bromine-containing kinase inhibitor H-89 was used to characterize discrete binding modes of the compound when complexed with the catalytic subunit of protein kinase A.
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.
protein kinase A; kinases; ligands; inhibitors; flexibility; anomalous dispersion; SAD; bromine
Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C4 dicotyledon that is closely related to Arabidopsis thaliana
Newell, Christine A.
Brown, Naomi J.
Hibberd, Julian M.
Journal of Experimental Botany
In leaves of most C4 plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C3 plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C4 pathway, and there are few genes that have been shown to be important for the cell biology and development of C4 leaves. To facilitate functional analysis of C4 photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C4 species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C4 species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of β-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C4 plant and should significantly accelerate the analysis of mechanisms underlying C4 photosynthesis.
Agrobacterium tumefaciens; Arabidopsis thaliana; C4 photosynthesis; Cleome gynandra; transformation
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