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Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate

Ruchira Engel1, Adrie H Westphal1, Daphne H E W Huberts1

  • 1Laboratory of Biochemistry, MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.

The Journal of Biological Chemistry
|July 22, 2008
PubMed

Insights

Macromolecular crowding compacts unfolded apoflavodoxin, increasing stability and secondary structure. However, it also causes aggregation of folding intermediates in vitro, necessitating chaperone assistance for efficient folding in vivo.

Area of Science:

  • Biochemistry
  • Protein Folding Dynamics
  • Cellular Biophysics

Background:

  • Understanding protein folding in vivo is crucial for cellular function.
  • Macromolecular crowding is a significant factor influencing protein conformation and stability within cells.
  • Apoflavodoxin exhibits a stable off-pathway intermediate with molten globule characteristics.

Purpose of the Study:

  • To investigate the effects of macromolecular crowding on the in vitro folding of apoflavodoxin.
  • To mimic cellular crowded conditions using dextran 20 and assess its impact on protein folding pathways.
  • To elucidate the role of crowding in protein stability, structure, and aggregation.

Main Methods:

  • Utilized dextran 20 (30% w/v) to simulate crowded cellular environments.
  • Employed fluorescence correlation spectroscopy to determine hydrodynamic radius.
  • Applied Förster resonance energy transfer and far-UV circular dichroism spectroscopy to measure protein volume, stability, and secondary structure changes.

Main Results:

  • Crowding by dextran 20 reduced apoflavodoxin's volume by approximately 29%, increasing stability by up to 1.1 kcal mol(-1).
  • Observed increased secondary structure and a 2.9°C rise in the midpoint of thermal unfolding for native apoflavodoxin.
  • Demonstrated severe aggregation of the off-pathway folding intermediate under crowded conditions in vitro.

Conclusions:

  • Macromolecular crowding compacts unfolded apoflavodoxin, restricting conformational sampling and potentially affecting folding kinetics.
  • Despite in vitro aggregation, apoflavodoxin folds efficiently in the cytoplasm of Escherichia coli, indicating the essential role of cellular chaperones (e.g., Trigger Factor, DnaK system).
  • Cellular machinery, particularly chaperones, is vital for overcoming crowding-induced challenges and ensuring proper protein folding in vivo.

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