Interdomain Linker Effect on the Mechanical Stability of Ig Domains in Titin

Figures

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  Figure 1. (a) Schematic architecture of one-half of the sarcomere highlights the polyprotein structure of giant protein titin (colored in purple), not in scale. (b) Structure shows human titin segment I(27–32) chosen for mechanical stability measurements. Except for I27 (PDB:1TIT), all others are simulated structures. (c) The cartoon shows the structure of I30 with a possible disulfide bond between Cys73 and Cys23, while no disulfide is present in other Ig domains. (d) The schematic shows how high-precision AFM-SMFS measures the mechanical stability of Ig domains with an artificial linker.

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  Figure 2. (a) Scheme of AFM-SMFS setup to measure the mechanical stability of polyprotein I(27–32). (b) Representative force-extension curves show the unfolding events of Ig domains (marked by a star). The top two curves are from the unfolding of polyprotein with an artificial 9G linker (colored in purple) and the bottom curves are from the natural polyprotein sequence without the linker. The I30 domain shows a peak with ΔLc of 11 nm (in orange), the remaining five Ig domains all show a peak with ΔLc of 28 nm, and GB1 shows a ΔLc of 18 nm. (c,d) Histograms show the unfolding force (c) and ΔLc (d) from the five Ig domains. (e) The histogram shows the ΔLc of I30.

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  Figure 3. (a) Scheme of AFM-SMFS setup to measure I(28–30) domains. (b) Representative force-extension curves from polyprotein with linkers (top, colored in blue) and without linker (bottom, black). (c,d) Histograms of their corresponding unfolding force (c) and ΔLc (d) are shown. The star indicates the unfolding event/peak of one protein domain.

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  Figure 4. (a) Scheme of AFM-SMFS setup to measure I(30–32) domains. (b) Representative force-extension curves from polyprotein with linkers (top, colored in green) and without linker (bottom, in black). (c,d) Histograms of their corresponding unfolding force (c) and ΔLc (d) are shown. The star indicates the unfolding event/peak.

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  Figure 5. (a–c) The scheme (top panel) and mechanical force of I30 in polyprotein I(27–32) (a), I(28–30) (b), and I(30–32) (c). The unfolding force histogram of I30 with the corresponding linker is shown in the middle panel, and the natural sequence without linker is shown at the bottom. The 9G linker was colored red, and the two cysteines forming the disulfide bond were depicted as orange spheres.