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.

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole, and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric oxide signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechano-chemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

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.

Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4–10 pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.

Magnetic tape heads are ubiquitously used to read and record on magnetic tapes in technologies as diverse as old VHS tapes, modern hard-drive disks, or magnetic bands on credit cards. Their design highlights the ability to convert electric signals into fluctuations of the magnetic field at very high frequencies, which is essential for the high-density storage demanded nowadays. Here, we twist this conventional use of tape heads to implement one in a magnetic tweezers design, which offers the unique capability of changing the force with a bandwidth of ∼10 kHz. We calibrate our instrument by developing an analytical expression that predicts the magnetic force acting on a superparamagnetic bead based on the Karlqvist approximation of the magnetic field created by a tape head. This theory is validated by measuring the force dependence of protein L unfolding/folding step sizes and the folding properties of the R3 talin domain. We demonstrate the potential of our instrument by carrying out millisecond-long quenches to capture the formation of the ephemeral molten globule state in protein L, which has never been observed before. Our instrument provides the capability of interrogating individual molecules under fast-changing forces with a control and resolution below a fraction of a piconewton, opening a range of force spectroscopy protocols to study protein dynamics under force. dynamic force spectroscopy | protein mechanics | protein folding | molten globule state | magnetic tape head M agnetic head recording systems have been perfected over decades, resulting in a deep understanding of the physical features of magnetic tape heads, which evolved to create strong magnetic fields that can change very rapidly in time (1, 2). Hence, it becomes natural that we explore the use of this technology in its application to force spectroscopy. Magnetic tweezers force spectroscopy uses magnetic field gradients to apply pulling forces on biomolecules tethered to superparamagnetic beads (3-9). Due to the extreme compliance of the magnetic trap, magnetic tweezers offer intrinsic force-clamp conditions and an exquisite control of the pulling force, which, combined with HaloTag covalent chemistry, provides an inherent stability and gives access to long timescales, of several hours or even days (10-13). However, in standard magnetic tweezers instruments, the force change is limited by the mechanical movement of the pair of magnets, which can take up to 100 ms, and impedes capturing early molecular events occurring upon fast force quenches or applying force protocols where the force changes rapidly, motivating the need to implement fast-changing magnetic fields.In this article, we present magnetic tape head tweezers, a force spectrometer capable of changing the force on a microsecond timescale, while maintaining an impeccable control of it. According to the Karlqvist description of the magnetic field created by a tape head (14), we provide a full analytic description of the pulling force, which allows us to calibrate our instrument over a ...