Individual globular domains and domain unfolding visualized in overstretched titin molecules with atomic force microscopy

Abstract

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin's globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme's ATP-binding site and hence contributing to the molecule's mechanosensory function.

Figure 1. Schematics of the titin-stretch experiment.

a. A titin molecule, attached by one of its ends (typically its M-line end, indicated with M) to the mica surface is pulled by a receding buffer droplet accelerated by centrifugal force (F_c). m is droplet mass and r is the distance from the center of rotation. b. At each time point during droplet movement, a surface-tension(γ)-based force (F_st, counteracted by the elastic force borne in the protein chain), proportional to chain diameter (D), stretches titin before it is stabilized by binding to the surface.

Figure 2. Titin molecules at different locations on the mica surface.

a. Place of sample application near the center of the mica surface. b. Edge of the sample application area. c. Surface near the edge of mica. Samples in Figs. a–c contained 1 M urea. d. Titin sample with no urea added. e. AFM image comparing the global structure of overstretched titin molecules in 0 mM (i) and 1 mM (ii) urea.

Figure 3. Length analysis of fully straightened and extended titin molecules.

a. High-resolution AFM image of a titin molecule. M and Z point at the globular heads in the respective sarcomeric locations, and the arrow indicates the direction of the receding meniscus. Inset, example of an overstretched titin molecule containing a large topographical gap that corresponds most plausibly to the PEVK domain (P). b. Topographical profile plot along the molecule’s axis indicating the globular heads. c. Length distribution of titin molecules (n = 255). Thick continuous line is a curve fit with the function , where A is frequency maximum, x₀ is length offset (1000 nm) and τ is decay constant. d. Examples of titin molecules stretched in the presence of 0.6 M KCl. P indicates the putative PEVK domain.

Figure 4. Topography analysis of fully straightened and extended titin.

a. Example of a stretched titin molecule with topographical gaps (G) highlighted. Arrow indicates the direction of the receding meniscus. Boxed area is analyzed in Fig. c. Inset, magnified and contrast-enhanced part of an overstretched titin molecule in which fine threads can be discerned in the topographical gaps (arrowheads). b. Cross-sectional topography of the extended titin molecule. Average peak height 1.4 Å, width at half maximum height 12.1 nm, and the corrected filament width (equation 1) is 9.3 nm (n = 183). The average cross-sectional profile was measured in a ∼50-nm-long filament region devoid of gaps. c. Axial height distribution of the molecule in the region boxed in Fig. a. Topographical gaps are indicated with G. d. Distribution of gap width (corrected according to equation 2). Mean gap width 27.7 nm (±23.8 nm S.D., n = 1879). Inset, gap-width distribution shown in logarithmic scale. e. Example of a conformationally relaxed titin molecule. C indicates coiled region of the molecule. f. Cross-sectional topography of the relaxed titin molecule in the region boxed in Fig. d. Peak height 3.5 Å, width at half maximum height 13.9 nm, and the corrected filament width (equation 1) is 9.6 nm (n = 51). g. Atomic force micrograph of titin recorded under aqueous buffer conditions. Height (i) and phase (ii) contrast images of the same scanned area are shown.

Figure 5. Folded globular domains in titin.

a–c. Examples of high-magnification AFM images in which ellipsoidal, globular structures can be identified along the contour of the titin molecule. d. Example of a topographical height profile along the axis of titin. e. Distribution of distance measured between consecutive topographical height peaks. Mean inter-peak distance 5.9 nm (±2.1 S.D., n = 325).

Figure 6. Analysis of domain unfolding near the M-line end of titin.

a. AFM images of overstretched titin molecules with the M-line end enlarged. M and G point at the globular M-line head of titin and the topographical gap corresponding to the most distal (i.e., farthest from the N-terminus) unfolded domain, respectively. 1 and 2 indicate the distance of the gap from the M-line center and the gap width, respectively. b. Distribution of the distance of the gap from the center of the M-line titin head. c. Distribution of gap width. d. Width of the distal gap as a function of end-to-end length. Linear fit, correlation coefficient (r) is 0.52. e. AFM image of a tiitn molecule stretched in the absence of urea. G indicates the most distal gap. f. Schematics of the molecular complex in the M-line (cf. [41]). Scale indicates distance, in nanometers, from the center of the M-line. g. Molecular model of the titin kinase domain (1TKI). The three N-terminal ß-strands (ßC1, ßC2, ßC3) are highlighted in yellow.