A protein’s folding time is the time required for it to reach its unique folded state starting from its unfolded ensemble. Globular, cytosolic proteins can only attain their intended biological function once they have folded. This means that protein folding times, which typically exceed the timescales of enzymatic reactions that proteins carry out by several orders of magnitude, are critical to determining when proteins become functional. Many scientists have worked tirelessly over the years to measure protein folding times, determine their theoretical bounds, and understand how they fit into biology. Here, I focus on one of the more interesting questions to fall out of this field over the years: how fast can a protein fold? Note that this is a very different question than asking “how fast do proteins fold?”
The folding times of many naturally occurring and engineered proteins have been measured; the Protein Folding Kinetics Database contains kinetic parameters for >100 individual proteins under standard conditions (Manavalan et al. Sci. Rep. 2019). A quick inspection of this database suggests that most proteins fold on timescales on the order of a millisecond, with a median of ~5 milliseconds for its 2-state folding proteins. This is how fast proteins tend to fold – but how fast can they fold?
In 2004, Kubelka, Hofrichter, and Eaton proposed that, based on both experimental and theoretical information, the “speed limit” of protein folding is set by N/100 μs, where N is the length of the protein (note that this only works with single-domain proteins). Villin headpiece, a 35-residue protein, was found to fold in 4 μs, which is quite close to its theoretical limit of 0.35 μs (Jan, Eaton, Hofrichter J. Mol. Biol. 2003). Based on the X-ray structure of villin (Thang et al. PNAS 2005), lysine was mutated to norleucine (which is a lysine without the terminal amino group on the sidechain) in order to remove two repulsive interactions thought to be slowing down its folding process. This new protein, which would come to be termed “super villin,” was found to fold in 0.7 μs, just ~2x the theoretical limit predicted for a protein its size (Eaton, J. Phys. Chem. B 2021).
To my knowledge, super villin remains the answer to the question of how fast a protein can fold. Much recent experimental and theoretical work has focused on further characterizing ultrafast folding proteins in terms of their transition paths and topology. If this quick story about protein folding kinetics caught your attention, consider reading William Eaton’s career retrospective in J. Phys. Chem. B (https://pubs.acs.org/doi/abs/10.1021/acs.jpcb.1c00206) which contains a wealth of references and information.
