The production and X-ray structure determination of perdeuterated Staphylococcal nuclease.
Staphylococcal Nuclease (SNase) has been chosen as a model protein system to evaluate the improvement in neutron diffraction data quality using fully perdeuterated protein. Large quantities of the protein were expressed in Escherichia coli grown in medium containing deuterated amino acids and deuterated water (D2O) and then purified. The mean perdeuteration level of the non-exchangable sites in the protein was found to be 96% by electrospray ionization mass spectrometry. The perdeuterated enzyme was crystallized and its X-ray structure determined. Crystals of perdeuterated SNase have been grown to 1.5 mm3. Crystallization conditions, space group and cell parameters were found to be the same for both native and perdeuterated forms of the protein. Comparison of these two forms of SNase revealed no significant structural differences between them at the atomic resolution of 1.9 A. Data collection using crystals of the perdeuterated protein is scheduled at the Brookhaven High Flux Beam Reactor.
Gamble
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TR
Graduate Group in Biophysics, University of California, San Francisco 94143-0448.
Clauser
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KR
Kossiakoff
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AA
D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D003903:Deuterium; D013058:Mass Spectrometry; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation
D003903:Deuterium; D008836:Micrococcal Nuclease