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1.
Fig. 4.

Fig. 4. From: Mini-thin filaments regulated by troponin-tropomyosin.

Mini-thin-filament particle length distribution. After size-exclusion chromatography, filaments were negatively stained and examined by electron microscopy. Particle lengths are shown as binned into 2-nm groups and expressed as percent total. The major peak corresponds to a Gaussian distribution of 45.7 ± 8.8 nm (SD).

Huiyu Gong, et al. Proc Natl Acad Sci U S A. 2005 January 18;102(3):656-661.
2.
Fig. 1.

Fig. 1. From: Mini-thin filaments regulated by troponin-tropomyosin.

SDS/PAGE of troponin and gelsolin–troponin fusion protein. Lane 1, bovine cardiac troponin prepared by reconstitution from isolated subunits TnT, TnI, and TnC followed by ion-exchange chromatography; lanes 2–3 (duplicates), similar to lane 1 except the reconstituted complex contained the gelsolin–TnT fusion protein instead of TnT; lane 4, native bovine cardiac troponin; Stnd, molecular weight standards. Results are from a 14% acrylamide gel.

Huiyu Gong, et al. Proc Natl Acad Sci U S A. 2005 January 18;102(3):656-661.
3.
Fig. 3.

Fig. 3. From: Mini-thin filaments regulated by troponin-tropomyosin.

Isolation of mini-thin filaments using size-exclusion chromatography. HR S500 chromatography (Lower) and gradient gel SDS/PAGE (Upper) of mini-thin filaments are shown. The minifilament peak is indicated on the chromatogram, performed with a column volume of ≈90 ml. Other peaks were variable in size from preparation to preparation and contained low protein concentrations. OD is in arbitrary units. Lanes 1–3 show column fractions on the leading edge, peak, and trail of the minifilament peak, respectively. pre, sample before column loading; Tm, tropomyosin.

Huiyu Gong, et al. Proc Natl Acad Sci U S A. 2005 January 18;102(3):656-661.
4.
Fig. 2.

Fig. 2. From: Mini-thin filaments regulated by troponin-tropomyosin.

Electron microscopy of mini-thin filaments. Conditions were as follows: 5 mM Mops, pH 7.0/2 mM MgCl2/100 mM KCl/0.2 mM ATP/0.2 mM DTT. Proteins were incubated in the following concentrations: 5 μM G-actin, 1.2 μM tropomyosin, 1 μM gelsolin–troponin, 1 μM tropomodulin, and 5 μM phalloidin. The samples were diluted 5-fold immediately before application to grids. (A) Tropomyosin was omitted. (B) Tropomodulin was omitted. (C) All components were present. (D) Higher magnification of minifilaments prepared with all components as in C and then chromatographed by using Sephacryl S-500 (see Materials and Methods).

Huiyu Gong, et al. Proc Natl Acad Sci U S A. 2005 January 18;102(3):656-661.
5.
Fig. 5.

Fig. 5. From: Mini-thin filaments regulated by troponin-tropomyosin.

Mini-thin filaments activate myosin S1 ATPase activity. Addition of mini-thin filaments to myosin S1 increased ATPase activity in a Ca2+-dependent manner, with a 2.6-fold overall activation by Ca2+ (filled circles), which indicates that short filaments, only one regulatory unit long, were sufficient for regulation. Each point corresponds to an average of three to five measurements. The results indicate a cooperative transition (solid line) with nH = 2.4 ± 0.5 and Ca2+ Kapp = 1.34 ± 0.14 × 106 m-1. Adjusted data for conventional thin filaments are shown also (open circles), after normalization and scaling to the same maximum and minimum rates (see Results).

Huiyu Gong, et al. Proc Natl Acad Sci U S A. 2005 January 18;102(3):656-661.
6.
Fig. 6.

Fig. 6. From: Mini-thin filaments regulated by troponin-tropomyosin.

Ca2+ binding to the regulatory sites of thin filaments and to regulatory sites of thin-filament particles. Ca2+ binding was monitored by titration of thin filaments (open circles) or minifilament particles (filled circles), in each case fluorescently labeled on cardiac TnC at Cys-84. Solid lines are best-fit curves and indicate a qualitatively different transition shape for the long thin filaments. Unlike the findings for thin-filament particles, the effect of Ca2+ on long thin filaments was cooperative, producing a steeper transition. Steadystate fluorescence results from three experiments are superimposed. Intensities were normalized to the fractional change produced by saturating Ca2+. The maximal fluorescence increase for short thin filaments was two-thirds of the maximal increase for conventional thin filaments (i.e., a 24% increase rather than 36% after Ca2+ addition). Ca2+ binding to the particles was slightly stronger (KCa2+ = 4.5 ± 0.2 × 106 m-1 for the particles vs. 2.78 ± 0.05 × 106 m-1 for thin filaments).

Huiyu Gong, et al. Proc Natl Acad Sci U S A. 2005 January 18;102(3):656-661.

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