Biology subjects

Eckels, E. C.

Publications and source records attributed to Eckels, E. C..

3 recordsLinked to original sources

bioRxiv2018

Disulfide bonds: the power switches of elastic proteins

The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between force and velocity of its constituent molecular systems. Here we demonstrate a switchable power amplifier in an Ig domain of the massive muscle protein titin. Titin is composed of many tandem repeats of individually foldable Ig domains, which unfold and extend during muscle stretch and readily refold when the force on titin is quenched during a contraction. Cryptic cysteine residues are common in elastic proteins like titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding domain. However, the functional significance of disulfide-bonds in titin Ig domains remains unknown and may be fundamental to muscle mechanics. Here we use ultra-stable magnetic tweezers force spectroscopy to study the elasticity of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time using both organic reagents and oxidoreductase enzymes. We show that presence of an oxidized disulfide bond allows the parent Ig domain to fold at much higher forces, shifting the midpoint folding probability from 4.0 pN to 12.8 pN after formation. The presence of disulfide bonds in titin regulates the power output of protein folding in an all-or-none manner, providing for example at 6.0 pN, a boost from 0 to 6,000 zeptowatts upon oxidation. At this same force, single molecular motors such as myosin are typically stalled and perform little to no work. We further demonstrate that protein disulfide isomerase (PDI) readily reintroduces disulfide bonds into unfolded titin Ig domains, an important mechanism for titin which operates under a resting force of several pN in vivo. Our results demonstrate, for the first time, the functional significance of disulfide bonds as potent power amplifiers in titin and provide evidence that protein folding can generate substantial amounts of power to supplement the myosin motors during a contraction.

biophysics↗

bioRxiv2018

A Magnetic Head Design for Manipulating Single Proteins with Force

Magnetic heads are ubiquitously used to record and read on magnetic tapes in technologies as diverse as old cassettes or VHS tapes, modern hard drive disks, or magnetic bands in credit/debit or subway cards. They are designed to convert electric signals into fluctuations on the magnetic field at very high frequencies, crucial for the high density storage which is demanded nowadays. Here, we twist this traditional use of magnetic heads and implement one in a new force spectrometer design, where the magnetic field is used to pull on proteins tethered to superparamagnetic beads. Our instrument offers the same features as magnetic tweezers--intrinsic force-clamp conditions, with accurate control of the force, and intrinsic stability--, but with the novel ability of changing the force instantaneously, which allows to investigate protein dynamics at very short timescales, or under arbitrary force signals. We calibrate our instrument by relying on Karlqvist approximation of the field created by a magnetic head--the first building block of magnetic recording theory--through the force scaling of the unfolding/folding step-sizes of protein L, used as a molecular template. This results in a force range between 0 and 50 pN, when working at distances above 250 m, and electric currents up to 1 A. We illustrate the potential of our instrument by studying the folding mechanism of protein L upon ultra-fast force quenches. This allows us to describe that, in a short timescale of 50 ms, the unfolded protein L evolves to an ensemble of weak collapsed states, eventually acquiring the native conformation in a timescale of seconds. Our instrumental development provides a unique capability of interrogating individual molecules under fast-changing force signals, which opens a range of novel force spectroscopy schemes of unexplored biological significance.

biophysics↗

bioRxiv2018

DsbA is a switchable mechanical chaperone

DsbA is a ubiquitous bacterial oxidoreductase that associates with substrates during and after translocation, yet its involvement in protein folding and translocation remains an open question. Here we demonstrate a redox-controlled chaperone activity of DsbA, on both cysteine-containing and cysteine-free substrate, using a magnetic tweezers-based single molecule force spectroscopy that enables independent measurements of oxidoreductase activity and chaperone behavior. Interestingly we found, this chaperone activity is tuned by the oxidation state of DsbA; oxidized DsbA is a strong promoter of folding, but the effect is weakened by reduction of the catalytic CXXC motif. We further localize the chaperone binding site of DsbA using a seven-residue peptide which effectively blocks the chaperone activity. We calculated that DsbA assisted folding of proteins in the periplasm generates enough mechanical work to decrease the ATP consumption needed for periplasmic translocation by up to 33%. In turn, pharmacologic inhibition of this chaperone activity may open up a new class of anti-virulence agents.

biophysics↗