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A Low-Dose Electron Diffraction Assay for Protection of Protein Structure against Damage from Drying

Published online by Cambridge University Press:  17 March 2004

William H. Massover
William H. Massover
Affiliation:
Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07101-1709, USA
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Abstract

A new assay using low-dose electron diffraction to measure the protection of protein structure against damage from drying is described. When thin single crystals of catalase are dried within water alone, low-dose electron diffraction yields no Bragg spots. Drying within an experimental aqueous solution that permits detection of diffraction spots thereby indicates a positive result, and the extent of these Bragg reflections into the high angle range gives a quantitative measure of the degree of protection. Bragg spots out to 3.7–3.9 Å are recorded for drying within 100 mM solutions of the known structure-preserving sugars, sucrose, tannin, and trehalose. The ability of trehalose to maintain native protein structure during drying starts between 10 and 25 mM, and changes only slightly at concentrations above this threshold; with drying in 150-mM trehalose, catalase crystals yield diffraction spots out to 3.7 Å. Drying within the organic nonsugar polymer polyvinylpyrrolidone gives Bragg spots to 4.0 Å. This new assay should be useful to measure the unexamined structure-preserving capabilities of modified sugars, other nonsugars, and mixtures to identify which protective matrix maintains native protein structure to the greatest extent during drying; electron crystallography using that optimal matrix should yield protein structure at improved levels of high resolution.

Keywords

catalaseelectron crystallographyelectron diffractionpolyvinylpyrrolidoneprotein structureresolutionsugar sustains
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 10 , Issue 2 , April 2004 , pp. 261 - 269
Copyright
© 2004 Microscopy Society of America

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