Measuring "unmeasurable" folding kinetics of proteins by single-molecule force spectroscopy.
Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to "un-boil" an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of "un-boiling eggs", providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.
Jollymore
Ashlee
A
Department of Chemistry, The University of British Columbia, Vancouver, BC, Canada.
Li
Hongbin
H
D001407:Bacillus / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D017510:Protein Folding; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D043364:Endo-1,4-beta Xylanases