Application of Dynamic Light Scattering in Protein Crystallization

Ariane Proteau1, Rong Shi1, Miroslaw Cygler2

1 Department of Biochemistry, McGill University, Montreal, Quebec, Canada, 2 Biotechnology Research Institute, Montreal, Quebec, Canada
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.10
DOI:  10.1002/0471140864.ps1710s61
Online Posting Date:  August, 2010
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Abstract

Success in determining the three‐dimensional structure of a macromolecule by X‐ray diffraction methods depends critically on the ability to obtain well ordered crystals of the macromolecule in question. Predisposition to crystallization correlates with the homogeneity of the molecules in solution. Dynamic light scattering (DLS) is particularly well suited for evaluating protein homogeneity under multiple conditions and at concentrations commensurate with crystallization conditions. This unit presents a typical protocol for DLS measurements of a protein sample, and describes approaches to improve protein homogeneity in solution. Curr. Protoc. Protein Sci. 61:17.10.1‐17.10.9. © 2010 by John Wiley & Sons, Inc.

Keywords: dynamic light scattering; crystallization of proteins; crystallography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: DLS Measurement of Protein Samples
  • Basic Protocol 2: DLS to Optimize Protein Solution Behavior for Crystallization
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DLS Measurement of Protein Samples

  Materials
  • Protein solution at appropriate concentration (see annotation to step 1)
  • 0.2‐ to 0.45 µm nitrocellulose filter disc (Whatman)
  • Refrigerated centrifuge
  • 96‐ or 384‐well flat‐bottom plastic plate (Thermo Scientific, part no. 95040000)
  • DynaPro plate reader with Dynamics software (Wyatt Technology Corporation; http://www.wyatt.com)

Basic Protocol 2: DLS to Optimize Protein Solution Behavior for Crystallization

  Materials
  • Protein sample, 1 to 2 mg/ml
  • Concentrated solutions of selected buffers and additives (Table 17.10.3), if possible, 10‐fold more concentrated than required for the experiments
  • Protein concentrator (e.g., Amicon centrifugal filter unit Ultra‐4)
  • 96‐ or 384‐well microtiter plate
  • Additional reagents and equipment for measuring DLS ( protocol 1)
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Figures

  •   FigureFigure 17.10.1 Correlation functions and the corresponding calculated histograms. Top row: experimental correlation function and the best calculated fit; bottom row: corresponding histograms of particle sizes. (A) Monomodal distribution; (B) Small percentage of aggregates; (C) Large aggregates.

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Literature Cited

Literature Cited
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   Bergfors, T. 1999. Dynamic light scattering. In Protein Crystallization: Techniques, Strategies, and Tips. A Laboratory Manual. (T. Bergfors, ed.) pp. 29‐38. International University Line, La Jolla, Calif.
   Borgstahl, G.E. 2007. How to use dynamic light scattering to improve the likelihood of growing macromolecular crystals. Methods Mol. Biol. 363:109‐29.
   Claes, P., Dunford, M., Kennedy, A., and Vardy, P. 1992. An on‐line dynamic light‐scattering instrument for macromolecular characterization. In Laser Light Scattering in Biochemistry (S.E. Harding, D.B. Sattelle, and V.A. Bloomfield, eds.) pp. 66‐76. Royal Society for Chemistry, Cambridge, U.K.
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   Zulauf, M. and D'Arcy, A. 1992. Light scattering of proteins as a criterion for crystallization. J. Crys. Growth 122:102‐106.
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