The Mechanical Power of Titin Folding

Figures

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  Figure 1.. Real-Time Control of Cryptic Disulfide Bonds in Single Polyproteins under Force (A) Under force, an Ig domain extends up to its buried disulfide bond, exposing it. Upon reduction, a further extension occurs. (B) The oxidation status and effect of cryptic disulfide bonds are evident in these two traces of polyproteins containing eight repeats of a titin Ig domain. Oxidized polyproteins extending at a high force of 84 pN increase their length in steps of 11 nm (blue trace), whereas reduced polyproteins extend in steps of 25 nm (red trace). While the rate of folding of the reduced polyprotein at 4 pN is slow, oxidation greatly accelerates the folding rate, driving it to completion at the same force. The increased folding probability at 4 pN is measured from the number of steps recovered with a probe pulse at 84 pN. (C) The status of a single polyprotein can be easily changed from oxidized to reduced by introducing a reducing agent into the solution (TCEP) while the polyprotein is fully unfolded, exposing its thiols to the solution. Step-size histograms record the changes observed from extending a fully oxidized polyprotein ± 2.1 nm), immediately after the addition of TCEP (14.6 ±…

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  Figure 2.. Cryptic Disulfide Bonds Alter the Folding Dynamics of Titin Ig Domains (A) (After full extension, oxidized polyproteins are allowed to fold by quenching the force to 10–16 pN for 100 s. While only a single folding event is observed at 16 pN, complete refolding occurs at 10.6 pN. (B) Reduced polyproteins fold at a much lower range of forces in the 2–5 pN range. (C) The folding probability is calculated as the number of refolded domains (probe pulse) divided by eight. Oxidation shifts the midpoint folding probability from 4.1 to 12.5 pN. The data were collected from several different molecules, with n = 11 for I27OXD and n = 9 for I27RED. (D) The folding steps observed during the quench pulse (insets in A and B) scale in size with the applied force. Oxidized folding steps (blue circles) are fit by the FJC model (solid blue line, see also equations in STAR Methods) using a contour length of 12.7 nm. Reduced folding steps (red circles) are fit using a much larger contour length of 28.4 nm (solid red line). These data came from the same molecules measured in (C).

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  Figure 3.. Oxidoreductase Mediated Disulfide Cleavage and Reformation under Force (A) After full unfolding of eight I27ox domains (11.4-nm steps), PDIRED was added to the flow cell, causing eight distinct disulfide cleavage events (14.6-nm steps). After quenching the force to 5 pN, a downward folding staircase is observed. Subsequent unfolding at 84 pN reveals a mixture of 11- and 14-nm steps, indicating reformation of the disulfide bond. (B) Disulfide reformation could also be achieved with oxidized PDI. After full unfolding of eight I27OXD domains, TCEP (10 mM) was added to the flow cell, resulting in eight 14.6-nm steps due to the reduction of the disulfide bond. Quenching the force to 5 pN resulted in only a single refolding event. The protein was stretched again at high force to remove the TCEP with 53 washes using buffer. Reducing the force to 2 pN caused the refolding of 73 × I27RED domains, as indicated by the 25-nm steps. After full unfolding, PDIOXD was added to the flow cell, and the force was reduced to 5 pN again. In the presence of the oxidoreductase, a folding staircase is again observed. Subsequent pulling at 84 pN demonstrates 73 × 11- and 14-nm steps, indicating nearly…

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  Figure 4.. PDI Introduces Disulfide Bonds after Accelerated Polypeptide Collapse (A) Folding trajectories of I27 in the presence of 60 mM PDIRED at 6.2, 5.2, and 4.3 pN. The extend and probe pulses contain both 11- and 14-nm steps due to the initial cleavage of the disulfide bond and subsequent oxidoreductase-mediated reformation of the disulfide bond during the quench. (B) Comparison of individual folding trajectories of I27RED in the presence of 10 mM TCEP versus I27OX after cleavage by 60 μM PDIRED. The greatly favored folded state in the presence of PDI suggests chaperone activity from the oxidoreductase. (C) The folding probability of I27 is shifted in favor of the folded state in the presence of PDI. The sigmoidal fits demonstrate a 1.0-pN shift of the midpoint folding probability from 4.0 pN in the presence of TCEP to 5.0 pN in the presence of PDIRED. (D) The folding step sizes observed in the presence of PDI closely followed the FJC model for the I27RED domain, even if there was disulfide refor-mation. This suggests that the oxidoreductase reintroduces the disulfide bond only after there is collapse of the polypeptide backbone.

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  Figure 5.. Oxidation Increases the Work Done by Protein Folding and Shifts Its Peak to Higher Forces We calculate the expected value of the work delivered by folding steps as the product of folding work times the folding probability. The expected value of the folding work delivered by a reduced Ig domain peaks at a force of 3.5 pN with a value of 25.6 zJ (solid red line), calculated by multiplying the reduced folding probability (empty red squares, dashed line) times the reduced folding work (empty red diamonds, dashed line). Oxidation causes sizeable effects on the mechanical work delivered by protein folding; the expected value of the folding work delivered by an oxidized Ig domain is now 2.5 times larger (63.9 zJ) and peaks at 11 pN (solid blue line); from the oxidized folding probability (empty blue diamonds, dashed line) times the oxidized folding work (empty blue squares, dashed line). However, in both cases, the delivery of the folding work at their peak is very slow due to measured folding kinetics.

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  Figure 6.. Oxidation Acts as a Power Switch Controlling the Delivery of Protein Folding Work (A) Force protocol for measuring fast folding contractions at forces at which the folding probability is always one. A fully extended oxidized polyprotein is first quenched to 16 pN, where no folding occurs. A rapid quench follows, resulting in a brief elastic recoil and a very fast folding contraction. A subsequent increase of the force up to 16 pN reveals a much shorter extension, marking the folding of all eight domains. The speed of the folding contraction increases greatly as the force of the quench is reduced. The folding contractions are fit with a single exponential to measure the folding rate. (B) The force dependency of the folding rate for oxidized domains (blue squares) and reduced domains (red squares). The dashed lines correspond to fits using Equation 1. (C) Force dependency of the initial velocity of the folding contraction (v0), measured as the amplitude of the contraction divided by its time constant (A, inset). The dashed lines correspond to fits from Equation 2. (D) The peak power delivered by the folding contraction is calculated as v0 · F. The dashed lines are fits of Equation…

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  Figure 7.. A PDI-Mediated Disulfide Switch of the Power Output from Titin Folding Upon initiation of a muscle contraction, the force on titin is lowered through engagement of the crossbridges. Titin domains lacking cysteines or containing reduced disulfide bonds are unable to fold rapidly and therefore contribute very little peak power, reaching a maximum of only 25 zW at a force of 2 pN (left). The oxidation of titin disulfide bonds through the PDI machinery of the cell allows titin domains to fold at higher forces and more rapidly. Despite the collapse step being much larger in the case of the reduced domain, the forces and kinetics from folding the oxidized domain at 4.0 pN are so much higher that the power output is two orders of magnitude greater.