Figure 1.
Spin labeling of a protein with the bi-functional spin label HO-1944.
HO-1944 attaches itself via two disulfide bonds to two cysteines separated by 4 residues.
Figure 2.
Circular Dichroism of the labeled Tm.
A. Mean residue ellipticity of the labeled Tm mutants, 13/17 (thin solid line), 153/157 (dashed line), 188/192 (dotted line), 268/272 (- . . -) and WT Tm (thick solid line). B. Magnified area of A.
Figure 3.
Thermal denaturation of labeled Tm.
Mean residue ellipticity of Tm 13/17 (filled square), 153/157 (open square), 188/192 (open circle), 268/272 (filled circle), and WT Tm (filled triangle) at 221 nm as a function of temperature.
Table 1.
Melting temperature and enthalpy of the labeled Tm.
Figure 4.
In-vitro motility of the labeled Tm.
Mean speed of regulated thin filaments reconstituted with WT Tm and the four labeled Tm: 13/17, 153/157, 188/192 and 268/272 under high calcium concentration, pCa 5 (open bar) and low calcium concentration, pCa 9 (filled bar).
Figure 5.
Dynamics of the labeled Tm in the muscle fiber.
A. Overlay of the cw-EPR spectra of labeled Tm in ghost fiber (black) and labeled Tm cross-linked to DITC glass beads in solution (blue). The double arrows correspond to the hyperfine splitting (2 Teff) of the cw spectra. B. Overlay of the ST-EPR spectra of labeled Tm in ghost fiber (black) and labeled Tm cross-linked to DITC glass beads in solution (blue). C. Comparison of the normalized first integral of the ST-EPR spectrum (V'2) of labeled Tm in ghost fiber (black) and labeled Tm cross-linked to DITC-glass beads (blue). The double arrows correspond to the height of the first integral of the normalized V'2 spectra.
Figure 6.
Dynamics of the labeled Tm in solution and in the muscle fiber.
Overlay of the cw-EPR spectrum of Tm in solution (black) versus Tm in the muscle fiber (red). The double arrow defines hyperfine splitting of the cw spectra.
Figure 7.
Gradient of Tm dynamics along its length.
Normalized first integral of V'2 spectra of 13/17 (white bar), 153/157 (black bar), 188/192 (red bar), and 268/272 (blue bar) in ghost fiber (A), in presence of Tn (B), in presence of Ca2+(C), and decorated with S1 (D).
Table 2.
Effective correlation time of the labeled Tm.