Single-molecule fluorescence studies of intrinsically disordered proteins

Methods Enzymol. 2010:472:179-204. doi: 10.1016/S0076-6879(10)72010-3.


Intrinsically disordered proteins (IDPs) (also referred to as natively unfolded proteins) play critical roles in a variety of cellular processes such as transcription and translation and also are linked to several human diseases. Biophysical studies of IDPs present unusual experimental challenges due in part to their broad conformational heterogeneity and potentially complex binding-induced folding behavior. By minimizing the averaging over an ensemble (which is typical of most conventional experiments), single-molecule fluorescence (SMF) techniques have recently begun to add advanced capabilities for structural studies to the experimental arsenal of IDP investigators. Here, we briefly discuss a few common SMF methods that are particularly useful for IDP studies, including SMF resonance energy transfer and fluorescence correlation spectroscopy, along with site-specific protein-labeling methods that are essential for application of these methods to IDPs. We then present an overview of a few studies in this area, highlighting how SMF methods are being used to gain valuable information about two amyloidogenic IDPs, the Parkinson's disease-linked alpha-synuclein and the NM domain of the yeast prion protein Sup 35. SMF experiments provided new information about the proteins' rapidly fluctuating IDP forms, and the complex alpha-synuclein folding behavior upon its binding to lipid and membrane mimics. We anticipate that SMF and single-molecule methods, in general, will find broad application for structural and mechanistic studies of a wide variety of IDPs, both of their disordered conformations, and their ordered ensembles relevant for function and disease.

MeSH terms

  • Amino Acids / chemistry
  • Fluorescence Resonance Energy Transfer / instrumentation
  • Fluorescence Resonance Energy Transfer / methods
  • Fluorescent Dyes / chemistry
  • Humans
  • Models, Molecular
  • Molecular Structure
  • Peptide Termination Factors / chemistry
  • Protein Binding
  • Protein Conformation*
  • Protein Denaturation
  • Protein Folding
  • Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Spectrometry, Fluorescence / instrumentation
  • Spectrometry, Fluorescence / methods*
  • Staining and Labeling / methods
  • alpha-Synuclein / chemistry


  • Amino Acids
  • Fluorescent Dyes
  • Peptide Termination Factors
  • Proteins
  • SUP35 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • alpha-Synuclein