Mechanical protein folding at the human time scale
The development of single molecule force-clamp spectroscopy introduces a novel way to probe the dynamics of proteins by measuring their length and mechanical stability during each stage of folding. Covalent tethering techniques in combination with drift-less Magnetic Tweezers have enabled single recordings that last over 10 hours. It should be feasible to hang on to the same molecule for weeks and monitor thousands of folding and unfolding transitions. Single-molecule techniques have always promised to capture rare events that can lead to misfolding diseases such as Alzheimer's and Parkinson's. Now, for the first time, we have the means to explore such rare and conformations in proteins.
In our experiments, we use the the force-quench technique to first unfold and extend a protein and then allow the protein to collapse and then fold by quenching the force to a lo value. This technique easily separates three distinct states during folding under force: the extended conformation,a weakly stable collapsed conformation corresponding to a "molten globule" and the native mechanically stable conformation. The collapse trajectory of an extended protein to its collapsed molten globule state is complex and governed by the recently discovered entropic barrier that forms when force is applied to a molecule. Once the molten globule state is reached, the protein needs to remain undisturbed for a surprisingly long time (seconds) to fully regain its native mechanically stable conformation. Mechanical stability is an excellent proxy for the distinct stages of folding. Our studies show that the physics of a folding protein can be accurately studied using force-clamp spectroscopy by AFM and MT. We are developing techniques that make possible to extract the free energy of a single protein from our experimental recordings. Thanks to our unique perspective on mechanical unfolding and folding of proteins, we are beginning to understand the mechanical architecture of adhesive pilins from Gram-positive bacteria.