Persistence length of titin from rabbit skeletal muscles measured with scattering and microrheology techniques

Abstract

The persistence length of titin from rabbit skeletal muscles was measured using a combination of static and dynamic light scattering, and neutron small angle scattering. Values of persistence length in the range 9-16 nm were found for titin-II, which corresponds to mainly physiologically inelastic A-band part of the protein, and for a proteolytic fragment with 100-nm contour length from the physiologically elastic I-band part. The ratio of the hydrodynamic radius to the static radius of gyration indicates that the proteins obey Gaussian statistics typical of a flexible polymer in a -solvent. Furthermore, measurements of the flexibility as a function of temperature demonstrate that titin-II and the I-band titin fragment experience a similar denaturation process; unfolding begins at 318 K and proceeds in two stages: an initial gradual 50% change in persistence length is followed by a sharp unwinding transition at 338 K. Complementary microrheology (video particle tracking) measurements indicate that the viscoelasticity in dilute solution behaves according to the Flory/Fox model, providing a value of the radius of gyration for titin-II (63 +/- 1 nm) in agreement with static light scattering and small angle neutron scattering results.

FIGURE 1

(a) Schematic arrangement of titin within the sarcomere, (b) diagram of titin structure, (c) electron micrograph of purified titin molecules, and (d) SDS-PAGE of titin preparation. (a) Single titin molecules span half of the sarcomere, with the N-terminus in the Z-line and C-terminus in the M-line. The A-band part of titin is bound to the thick/myosin filament and the I-band part forms an elastic connection between the tip of the thick filament and the Z-line. (b) The A-band part of titin (shown in black) contains both Ig and Fn3 domains; the elastic I-band part is formed by two segments of Ig domains arranged in tandems (medium gray) that are separated by unique sequences (light gray). The position of the proteolytical fragment that was isolated from the “elastic” part of titin is marked (Fr). (c) The contour length of the purified molecules is ∼1 μm. The C-terminals have characteristic small “heads”. (d) Left column shows sarcomere proteins as molecular weight markers. The titin preparation (right column) contains >90% of titin, most of which is in titin-II or β-connectin form.

FIGURE 2

Representative SLS curve (I(q) versus q) for titin in buffer A. The solid line is the fit to the data according to Eq. 1a.

FIGURE 3

(a) The temperature dependence of the radius of gyration for titin-II in buffer B measured with static light scattering from fits to Eq. 1a. The arrow indicates the point at which the protein is denatured. (b) The temperature dependence of the persistence length from DLS/SLS measurements of titin-II in buffer B; ○ (Eq. 1b), • (Eq. 8).

FIGURE 4

Relaxation spectra from Contin analysis for titin-II in buffer B. Data refer to a representative scattering angle ϑ of 90°, at T = 298 K.

FIGURE 5

The concentration dependence of the diffusion coefficient (D) for titin-II in buffer A. Data refer to uncentrifuged solutions.

FIGURE 6

DLS normalized correlation functions g₂(q,t) from titin-II (c = 1.41 mg/mL) and the fragment from the I-band part of titin (c = 1.61 mg/mL) (inset) in buffer B. Data refer to a range of different temperatures (298–333 K) and scattering angle of 90°.

FIGURE 7

The temperature dependence of the hydrodynamic radius: titin-II (○), and the fragment from the I-band part of titin (•). The arrow indicates the denaturated state of the protein. Both the data sets refer to preparations in buffer B.

FIGURE 8

(a) SANS profiles (dΣ/dΩ vs. q) of titin-II (c = 1.31 mg/mL) in deuterated buffer A. The solid line indicates the fit of the experimental data to Eq. 9. The inset shows a Kratky-Porod representation, highlighting the crossover between rigid chain conformations (I ∼ q⁻¹) at small length scales and Gaussian chains conformations (I ∼ q⁻²) at large length scales. (b) SANS profiles (dΣ/dΩ vs. q) of titin fragment from the I-band part of the molecule (c = 1 mg/mL) in deuterated buffer B. The solid line indicates the fit of the experimental data to Eq. 9.

FIGURE 9

Guinier plot (ln dΣ/dΩ vs. q²) of the low q section of the SANS data from titin in deuterated buffer A. The fit of the experimental data to Eq. 6 provides a value for R_g of 56 ± 5 nm.

FIGURE 10

Mean-square displacement of poly(amino) beads from particle tracking microrheology as a function of time at a series of titin-II concentrations in buffer A for a range of concentrations between 0.12 and 0.50 mg/mL. The linear dependence indicates diffusion in a purely viscous solvent, i.e., 〈Δr²〉 = 4 Dt.

FIGURE 11

The intrinsic viscosity of the titin-II as a function of polymer concentration. The solid line indicates the best linear fit to the data according to Eq. 12.

FIGURE 12

Schematic diagram of a section of titin molecule in a pore formed by hexagonally packed actin/thin filaments, i.e., near the I-/A-boundary of sarcomere beyond the myosin/thick filament. The blob concept is used to calculate the effect of the steric interactions with the actin filaments on the extension and entropic force of the chain.