Abstract
In this review, I discuss the various methods researchers use to unfold proteins in the lab in order to understand protein folding both in vitro and in vivo. The four main techniques, chemical-, heat-, pressure- and force-denaturation, produce distinctly different unfolded conformational ensembles. Recent measurements have revealed different folding kinetics from different unfolding mechanisms. Thus, comparing these distinct unfolded ensembles sheds light on the underlying free energy landscape of folding.
Highlights
Ever since Anfinsen discovered that a protein can be reversibly folded and unfolded outside of a cell[1], researchers have been investigating the folding process in vitro, confident in the knowledge that they were trying to understand a physical process of how the polypeptide chain finds a lowest free energy state
As the descriptions above have made clear, each of these unfolding mechanisms produces an ensemble of conformations that is distinct from the others
In collaboration with Bill Eaton’s lab at the NIH, demonstrated that the folding rates of the villin headpiece subdomain (HP35), one of the fastest known folders, are 5-fold slower for folding after dilution of denaturant compared to laser temperature jump (T-jump)[67]
Summary
Ever since Anfinsen discovered that a protein can be reversibly folded and unfolded outside of a cell[1], researchers have been investigating the folding process in vitro, confident in the knowledge that they were trying to understand a physical process of how the polypeptide chain finds a lowest free energy state. The kinetics of unfolding are typically extremely slow, orders of magnitude slower than unfolding with denaturant or temperature[48,49] This allows the use of careful NMR measurements to map which residues change structure first and can sometimes find complex folding dynamics. This unfolding mechanism is well understood physically, though the time-range over which these experiments are typically performed (10–100 nm/s stretching speeds) is somewhat longer than typical simulation times The appeal of these types of measurements is two-fold: 1) they are naturally single-molecule measurements, allowing the researcher to explore heterogeneity in folding pathways[62,63,64,65], and 2) the unfolded conformation eventually reached is very well defined, a completely extended polymer[65]. If folding is induced at a low force, the instrument typically has no resolution to observe the event
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