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-micron 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.

Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin Ig domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6-8 pN. We use single molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but so far unrecognized contributor to the force generated by a contracting muscle.

The mechanical stability of force-bearing proteins is crucial for their functions. However, slow transition rates of complex protein domains have made it challenging to investigate their equilibrium force-dependent structural transitions. Using ultra stable magnetic tweezers, we report the first equilibrium single-molecule force manipulation study of the classic titin I27 immunoglobulin domain. We found that individual I27 in a tandem repeat unfold/fold independently. We obtained the force-dependent free energy difference between unfolded and folded I27 and determined the critical force (∼5.4 pN) at which unfolding and folding have equal probability. We also determined the force-dependent free energy landscape of unfolding/folding transitions based on measurement of the free energy cost of unfolding. In addition to providing insights into the force-dependent structural transitions of titin I27, our results suggest that the conformations of titin immunoglobulin domains can be significantly altered during low force, long duration muscle stretching.

Here we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance, and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting proteinprotein interactions.

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an AFM cantilever and a glass surface using HaloTag anchoring at one end, and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ~2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a four-fold increase, from 131 pN to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice more sensitive to pulling force compared to protein L, and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.