High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase. - Archive ouverte HAL Access content directly
Journal Articles Acta crystallographica Section D : Structural biology [1993-...] Year : 2005

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Abstract

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

Dates and versions

hal-00187549 , version 1 (14-11-2007)

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Isabelle Hazemann, Marie-Thérèse Dauvergne, Matthew P. Blakeley, Flora Meilleur, Michael Haertlein, et al.. High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.. Acta crystallographica Section D : Structural biology [1993-..], 2005, 61 (Pt 10), pp.1413-7. ⟨10.1107/S0907444905024285⟩. ⟨hal-00187549⟩
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