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Biophysical Reviews

, Volume 5, Issue 2, pp 137–145 | Cite as

Effects of macromolecular crowding agents on protein folding in vitro and in silico

  • Alexander Christiansen
  • Qian Wang
  • Margaret S. Cheung
  • Pernilla Wittung-StafshedeEmail author
Review

Abstract

Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects—present when polypeptides normally fold in vivo—modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton’s early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i.e., less misfolding and/or kinetic heterogeneity) than in buffer.

Keywords

Protein folding Macromolecular crowding Spectroscopy Protein stability Excluded volume Coarse-grained simulation 

Notes

Acknowledgements

We would like to thank Allen Minton for his continuous (careful and sometimes critical) feedback on our work on crowding. Minton’s pioneering theoretical contributions to this field has served as the basis for many of our studies and they continue to stimulate new experiments. We thank Eefei Chen, University of California, Santa Cruz for preparing Fig. 3 and Jörgen Åden, Umeå University for preparing Fig. 1; and Magnus Wolf-Watz, Umeå University for helpful comments on the text.

Conflict of interest statement

The authors declare that they have no conflict of interest.

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Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Alexander Christiansen
    • 1
  • Qian Wang
    • 2
  • Margaret S. Cheung
    • 2
  • Pernilla Wittung-Stafshede
    • 1
    Email author
  1. 1.Department of ChemistryUmeå UniversityUmeåSweden
  2. 2.Department of PhysicsUniversity of HoustonHoustonUSA

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