Strong binding of myosin heads stretches and twists the actin helix

Abstract

Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by 0.22% when relaxed fibers were put into low-tension rigor (<10 kN/m(2)) demonstrating that strongly bound myosin heads elongate the actin filaments even in the absence of external tension. The pitch of the 5.9-nm actin layer line increased by approximately 0.62% and that of the 5.1-nm layer line decreased by approximately 0.26%, suggesting that the elongation is accompanied by a decrease in its helical angle (approximately 166 degrees) by approximately 0.8 degrees. This effect explains the difference between actin compliance revealed from mechanical experiments with single fibers and from x-ray diffraction on whole muscles. Our measurement of actin compliance obtained by applying tension to fibers in rigor is consistent with the results of mechanical measurements.

FIGURE 1

X-ray diffraction patterns collected in relaxed (upper half) and low-tension rigor (lower half) states from 17 muscle fiber bundles. The equator is vertical. White corresponds to higher intensity and a logarithmic gray scale is used. The center of the patterns appears overexposed to bring out the weaker features toward the edge of the patterns. The black rectangle in the center of the pattern is a metal beam stop to shield the detector from the direct x-ray beam. For each state only one-half of the averaged pattern is shown. The 5.9-nm, 5.1-nm, and 2.73-nm actin reflections are marked by arrows. White rectangles show the area of integration along the reciprocal radius used for calculation of the spacing of the reflections (Figs. 2 and 3). The total exposure of the bundles was 9.5 s in the relaxed state and 6.7 s in rigor. Rigor tension was <10 kN/m².

FIGURE 2

Meridional profile in the region of the 2.73-nm meridional reflection in the relaxed state (thin line) and in rigor (thick line). (A) Same data as in Fig. 1 integrated along the reciprocal radius from −0.013 nm⁻¹ to +0.013 nm⁻¹ (Fig. 1), background subtracted. (B) Same profiles as in A normalized for the peak intensity of the 2.73-nm reflection. Black dotted lines in B show the Gaussian fits of the upper halves of the peaks.

FIGURE 3

Meridional profiles of the 5.1-nm and 5.9-nm actin layer lines in the relaxed state (thin line) and in rigor (thick line). Panels A and C and B and D correspond to the left and right sides of the pattern (Fig. 1), respectively. (A and B) Same data as in Fig. 1 integrated along the reciprocal radius R from 0.02 nm⁻¹ to 0.04 nm⁻¹, background intensity subtracted. (C and D) Same profiles as in A and B normalized for the intensity of the peaks of the 5.1-nm and 5.9-nm layer lines, respectively. Open and closed symbols in C and D represent data points for relaxed and rigor states, correspondingly; solid lines are the Gaussian fits to the upper halves of the peaks.

FIGURE 4

Protocol used for measurement of actin compliance in rigor. From top to bottom: an example of records of change in the length of the bundle of muscle fibers, rigor tension, and signal from a pin photodiode showing times of opening of the x-ray shutter.

FIGURE 5

X-ray diffraction patterns collected from four bundles of muscle fibers in rigor using the protocol shown in Fig. 4. Upper and lower halves of the patterns correspond to low- and high-tension rigor, respectively. Total exposure in each state was 2.3 s.

FIGURE 6

Meridional profiles of the 2.73-nm actin meridional reflection in low- and high-tension rigor (thin and thick lines, respectively). (A) Same data as in Fig. 5, same integration range as in Fig. 2. (B) Profiles of the peaks normalized for maximal intensity; dotted lines show the Gaussian fits to the upper halves of the peaks.

FIGURE 7

Meridional profiles of the 5.1-nm and 5.9-nm actin layer lines in low- and high-tension rigor (thin and thick lines, respectively). Panels A and C and B and D correspond to the left and right sides of the pattern (Fig. 5), respectively. (A and B) Same data as in Fig. 5, integrated along the reciprocal radius R from 0.02 nm⁻¹ to 0.04 nm⁻¹, background intensity subtracted. (C and D) Same profiles as in A and B normalized for the intensity of the peaks of the 5.1-nm and 5.9-nm layer lines, respectively. Open and closed symbols in C and D represent data points for low- and high-tension rigor, correspondingly; solid lines are the Gaussian fits to the upper halves of the peaks.