Single-molecule fluorescence studies of protein folding.
The structural and dynamic details of protein folding are still widely unexplored due to the enormous level of heterogeneity intrinsic to this process. The unfolded polypeptide chain can assume a vast number of possible conformations, and many complex pathways lead from the ensemble of unfolded conformations to the ensemble of native conformations in an overall funnel-shaped energy landscape. Classical experimental methods involve measurements on bulk samples and usually yield only average values characteristic of the entire molecular ensemble under study. The observation of individual molecules avoids this averaging and allows, in principle, microscopic distributions of conformations and folding trajectories to be revealed. Fluorescence-based techniques are arguably the most versatile single-molecule methods at present, and Förster resonance energy transfer (FRET) between two dye molecules specifically attached to the protein of interest provides a means of studying the inter-dye distance and, thereby, the conformation of folding polypeptide chains in real time. This chapter focuses on practical aspects and different experimental realizations for protein folding investigations by using single-molecule fluorescence.
Nienhaus
G Ulrich
GU
Institute of Biophysics, University of Ulm, Ulm, Germany.
D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D008856:Microscopy, Fluorescence; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D011506:Proteins