European Biophysics Journal

, Volume 25, Issue 3, pp 211–219

Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding

  • K. Gast
  • Angela Nöppert
  • Marlies Müller-Frohne
  • Dietrich Zirwer
  • Gregor Damaschun
ARTICLE

DOI: 10.1007/s002490050033

Cite this article as:
Gast, K., Nöppert, A., Müller-Frohne, M. et al. Eur Biophys J (1997) 25: 211. doi:10.1007/s002490050033

Abstract

Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 °C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available.

Key words Protein folding Dynamic light scattering Stopped-flow kinetics Ribonuclease A Phosphoglycerate kinase 

Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • K. Gast
    • 1
  • Angela Nöppert
    • 1
  • Marlies Müller-Frohne
    • 1
  • Dietrich Zirwer
    • 2
  • Gregor Damaschun
    • 2
  1. 1.Max-Delbrück-Center for Molecular Medicine Berlin-Buch, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany (Fax: +49 30 9406 2548; e-mail: gastorion.rz.mdc-berlin.de)DE
  2. 2.Humboldt University at Berlin, Institute of Biology, c/o Max-Delbrück-Center for Molecular Medicine Berlin-Buch, Robert-Rössle-Strasse 10, D-13122 Berlin, GermanyDE

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