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Nat Methods. 2015 Sep;12(9):859-65. doi: 10.1038/nmeth.3493. Epub 2015 Aug 3.

ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.

Author information

1
Research Group of 3D Electron Cryomicroscopy, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
2
Department of Biochemistry, Theodor-Boveri Institute, University of Würzburg, Würzburg, Germany.
3
Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany.
4
Institut für Zoologie - Abteilung für Molekular Tierphysiologie, Johannes Gutenberg Universität Mainz, Mainz, Germany.
5
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
6
Research Institute of Molecular Pathology, Vienna, Austria.
7
Department for Bioanalytics, Georg-August University Göttingen, Göttingen, Germany.
8
Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Abstract

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.

PMID:
26237227
PMCID:
PMC5136620
DOI:
10.1038/nmeth.3493
[Indexed for MEDLINE]
Free PMC Article

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