12 research outputs found
The Mechanical Power of Titin Folding
Summary: The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band. We find that formation of the disulfide regulates the peak power output of protein folding in an all-or-none manner, providing at 6.0 pN, for example, a boost from 0 to 6,000 zW upon oxidation. This mechanism of power generation from protein folding is of great importance for muscle, where titin domains may unfold and refold with each extension and contraction of the sarcomere. : Eckels et al. use single-molecule magnetic tweezers to simultaneously probe the folding dynamics of titin Ig domains and monitor the redox status of single disulfides within the Ig fold. Oxidation of the disulfide bond greatly increases both the folding force and the magnitude of power delivered by protein folding. Keywords: protein folding, titin, single molecule, magnetic tweezers, force spectroscopy, disulfide bond, mechanical power, muscle contraction, oxidative folding, oxidoreductas
Work Done by Titin Protein Folding Assists Muscle Contraction
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 immunoglobulin (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 as yet unrecognized, contributor to the force generated by a contracting muscle
Direct Observation of Titin Immunoglobulin Domain Unfolding-Refolding in Muscle Sarcomeres
Ephemeral states in protein folding under force captured with a magnetic tweezers design
Proteins Breaking Bad: A Free Energy Perspective
Protein aging may
manifest as a mechanical disease that compromises
tissue elasticity. As proved recently, while proteins respond to changes
in force with an instantaneous elastic recoil followed by a folding
contraction, aged proteins <i>break bad</i>, becoming unstructured
polymers. Here, we explain this phenomenon in the context of a free
energy model, predicting the changes in the folding landscape of proteins
upon oxidative aging. Our findings validate that protein folding under
force is constituted by two separable components, polymer properties
and hydrophobic collapse, and demonstrate that the latter becomes
irreversibly blocked by oxidative damage. We run Brownian dynamics
simulations on the landscape of protein L octamer, reproducing all
experimental observables, for a naive and damaged polyprotein. This
work provides a unique tool to understand the evolving free energy
landscape of elastic proteins upon physiological changes, opening
new perspectives to predict age-related diseases in tissues
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