Watching conformational- and photo-dynamics of single fluorescent proteins in solution

Nat Chem. 2010 Mar;2(3):179-86. doi: 10.1038/nchem.545.

Abstract

Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the Anti-Brownian ELectrokinetic (ABEL) Trap, which allows extended investigation of solution-phase biomolecules - without immobilization -through real-time electrokinetic feedback. Here we apply the ABEL trap to study an important photosynthetic antenna protein, Allophycocyanin (APC). The technique allows the observation of single molecules of solution-phase APC for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation, and biomaterials for solar energy harvesting.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrodes
  • Immobilized Proteins / chemistry*
  • Immobilized Proteins / metabolism*
  • Light*
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / metabolism*
  • Microfluidic Analytical Techniques
  • Models, Molecular
  • Osmosis
  • Photobleaching
  • Phycocyanin / chemistry*
  • Phycocyanin / metabolism*
  • Protein Conformation / radiation effects
  • Protein Multimerization
  • Solutions
  • Spectrometry, Fluorescence
  • Time Factors

Substances

  • Immobilized Proteins
  • Luminescent Proteins
  • Solutions
  • allophycocyanin
  • Phycocyanin