Production and preliminary analysis of perdeuterated yeast inorganic pyrophosphatase crystals suitable for neutron diffraction.
Yeast inorganic pyrophosphatase (Y-PPase) is a model system for studying phosphoryl-transfer reactions catalysed by multiple metal ions. To understand the process requires knowledge of the positions of the protons in the active site, which can be best achieved by neutron diffraction analysis. In order to reduce the hydrogen incoherent-scattering background and to improve the signal-to-noise ratio of the neutron reflections, deuterated protein was produced. Deuterated protein 96% enriched with deuterium was produced in high yield and crystals as large as 2 mm on one side were obtained. These crystals have unit-cell parameters a = 58.9, b = 103.9, c = 117.0 A, alpha = beta = gamma = 90 degrees at 273 K and diffract neutrons to resolutions of 2.5-3 A. The X-ray structure of the perdeuterated protein has also been refined at 273 K to 1.9 A resolution.
Turku Centre for Biotechnology, University of Turku and Abo Akademi University, PO Box 123, FIN-20521 Turku, Finland.
Vesa U
VU
Tuominen
Dean A A
DA
Myles
Marie-Thérèse
MT
Dauvergne
Reijo
R
Lahti
Pirkko
P
Heikinheimo
Adrian
A
Goldman
D003460:Crystallization; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry*; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification*
D008670:Metals; D010710:Phosphates; D029701:Saccharomyces cerevisiae Proteins; D003903:Deuterium; D043564:Inorganic Pyrophosphatase