Disulfide isomerization reactions in titin immunoglobulin domains enable a mode of protein elasticity

Fig. 1. Conserved cysteine positions in the I65–I70 fragment of titin. a Schematic architecture of one half of the sarcomere. The titin filament spans across the contractile unit and its elastic properties arise from its extensible I-band region where Ig modules (filled black circles) can mechanically unfold and refold. The two entropic springs N2B and PEVK are also depicted as unstructured chains. Gray circles represent the repetition of Ig and fibronectin domains (FnIII) in the A- and M-band. b Crystal structure of the rabbit titin segment I65–I70 (pdb: 3B43). This 566 amino acid-long portion of proximal titin is organized in six tandem Ig modules represented in gray. Cysteines are marked by spheres, which are colored according to their position in the Ig secondary structure topology. Cysteines in β-strands B, F and G are shown in yellow, blue and red, respectively. c Scheme of the topology of a typical Ig domain of titin50, which displays the arrangement of the three clustered cysteines CysB, CysF and CysG. CysF and CysG appear in two adjacent β-strands whereas CysB is located in the opposite β-sheet (upper plane)

Fig. 2. Disulfide bonds in I65–I70. a Schematic representation of cysteine position in the six Ig domains composing I65–I70, and X-ray structure of I67 (pdb: 3B43) with arrows representing the applied force at the termini in AFM experiments. CysB, CysF and CysG are highlighted. Step sizes are indicated in the diagrams. We fitted Gaussian curves to all histograms presented here to estimate the different populations of observed step sizes. b Typical trace recorded applying a constant force of 170 pN on I65–I70reduced. c The histogram of step sizes shows that only reduced domains are detected (unfolding step of 26 nm). d Stretching I65–I70oxidized at 100 pN produces new distinct populations of shorter steps, with a different kinetics of appearance. The first 3 s of the experiment are shown in red. In this experimental recording, the polyprotein extends over time in steps of 26, 10, 6, 6 and 10 nm and in a last 4 nm step (in light gray, a 15 s portion of the trace has been cut out). e Histograms of the step sizes obtained with I65–I70oxidized (red in the first 3 s and gray for delayed events). The I65–I70oxidized unfolds showing two large populations of 6 and 10 nm steps in agreement with…

Fig. 3. Mechanical characterization of I69oxidized identifies disulfide isomerization as a contributor to titin elasticity. Diagrams on the left describe the mechanism of extension of I69 due to unfolding a, b and unfolding-triggered redox reactions c, d. Each pathway is illustrated with a trace and a histogram of step sizes. Theoretical step sizes are indicated by dashed lines (Supplementary Note 2). a The unfolding of (I69reduced)8 pulled at 170 pN results in a uniform step-wise extension of the polyprotein (26 nm steps in the blue trace). Similar to the trace shown in Fig. 2f, the absence of steps in the probe pulse indicates low refolding kinetics of I69reduced (Δt = 5 s). b When (I69oxidized)8 is stretched at 100 pN, a predominant population of 6 nm emerges in the first 3 s, marking the unfolding of I69 domains containing disulfide CysB–CysG. c After 3 s (gray part of the trace in (b)), two new populations of 4 and 17 nm steps appear. Mechanical unfolding activates a reaction of disulfide exchange where the thiol group of CysF can attack either the sulfur of CysB or CysG, giving rise to the 4 and 17 nm step populations. d In the presence of 10 mM Tcep, additional 19 nm steps are…

Fig. 4. Disulfide bonds decrease the mechanical stability of titin Ig domains. a Typical unfolding traces of (I69)8 recorded with a linear force increase of 40 pN s−1. Blue asterisks mark the unfolding events of I69reduced (26 nm steps in the blue upper trace) whereas red asterisks correspond to unfolding steps of I69oxidized (6 nm steps in the red lower trace). Isomerization reactions are detected as additional steps (gray triangles). b Unfolding forces and step sizes are represented in a bidimensional histogram. Average values appear as white crosses

Fig. 5. Disulfide bonds enable refolding of I69. a We use the engineered heteropolyprotein (I91ΔCys)2–I69-(I91ΔCys)2 to characterize the influence of disulfide bonds on the refolding of I69oxidized (quench Δt = 10 s, unfolding/probe force = 130 pN). Only traces that show the same extension (doted gray lines) at the end of the unfolding and probe pulses (before isomerization steps in the probe pulse) are considered. This recording indicates that I69oxidized unfolds in the unfolding pulse (6 nm step) and refolds during the quench while the disulfide CysB–CysG remains formed (6 nm step in the probe pulse). In this example trace, a subsequent isomerization step is detected in the probe pulse (solid triangle). b In this trace, disulfide CysB–CysF is formed through isomerization during the unfolding pulse (4 nm step, gray empty triangle). A 10 nm step in the probe indicates that I69 has refolded during the quench pulse keeping disulfide CysB–CysF. c Alternatively, if CysF–CysG forms (17 nm step, gray filled triangle), the absence of 23 nm step in the probe suggests an inability to refold during the quench. d Histogram summarizing how the three possible disulfide bonds formed within the triad…

Fig. 6. Disulfide isomerization in the Ig domains of titin. a Conservation of cysteines in the 163 Ig domains of the canonical titin. The upper histogram displays the cysteine percent conservation in the 101 Ig modules located in the I-band of titin. The lower histogram presents the percent conservation of cysteines in the 62 Igs in non-elastic regions (Z-Disk, A-Band and M-Band). The most conserved cysteines CysB, CysF and CysG, are shown in yellow, blue and red, respectively, and are over represented specifically in the I-band. b Mapping of cysteines in the structure of Ig domains of the I-band of titin. This 3D map is obtained by superimposition of the 101 Ig structures (X-ray and homology models) belonging to the I-band. For clarity, only the structure of I91 is represented. Each cysteine is marked at its Cβ atom by a colored dot for the cysteines CysB (yellow, C23’), CysF (blue, C73’) and CysG (red, C80’) or a black dot for other cysteine positions. c Left: Each bar marks the position along the canonical titin sequence of domains with specific distributions of CysB, CysF and CysG. These domains are concentrated in the I-band. Cardiac titin filaments are expressed in two major…