A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics

Under
physiological conditions, protein oxidation and misfolding
occur with very low probability and on long times scales. Single-molecule
techniques provide the ability to distinguish between properly folded
and damaged proteins that are otherwise masked in ensemble measurements.
However, at physiological conditions these rare events occur with
a time constant of several hours, inaccessible to current single-molecule
approaches. Here we present a magnetic-tweezers-based technique that
allows, for the first time, the study of folding of single proteins
during week-long experiments. This technique combines HaloTag anchoring,
sub-micrometer positioning of magnets, and an active correction of
the focal drift. Using this technique and protein L as a molecular
template, we generate a magnet law by correlating the distance between
the magnet and the measuring paramagnetic bead with unfolding/folding
steps. We demonstrate that, using this magnet law, we can accurately
measure the dynamics of proteins over a wide range of forces, with
minimal dispersion from bead to bead. We also show that the force
calibration remains invariant over week-long experiments applied to
the same single proteins. The approach demonstrated in this Article
opens new, exciting ways to examine proteins on the “human”
time scale and establishes magnetic tweezers as a valuable technique
to study low-probability events that occur during protein folding
under force.