In Situ Orientations of Protein Domains Roisean E. Ferguson, Yin-Biao Sun, Pascal Mercier, Andrew S. Brack, Brian D. Sykes, John E.T. Corrie, David R. Trentham, Malcolm Irving  Molecular Cell  Volume 11, Issue 4, Pages 865-874 (April 2003) DOI: 10.1016/S1097-2765(03)00096-0 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 Bifunctional Rhodamine Labeling of TnC (A) Arrangement of tropomyosin and the troponin components TnT, TnI, and TnC on the actin filament, modified from Heeley et al. (1987). (B) Ribbon representation of avian fast skeletal muscle TnC (Herzberg and James, 1988) showing the four pairs of cysteine residues (β-carbons shown as yellow spheres) that were crosslinked in a series of mutant TnCs by the BR probe. Ca2+ ions are shown as gray spheres. The N lobe of TnC is in the APO state. Residue 63 on the C helix that was replaced by a cysteine is not visible in this view. (C) Ribbon representation of the N lobe of avian fast skeletal muscle TnC where the N lobe is in the apo- (left panel) and Ca2+-bound state (right panel) (from Gagné et al., 1995). The view is down the D helix from V83 to E76. Modeled using ViewerPro (Accelrys Inc., San Diego, CA). (D) Bis-iodoacetamidorhodamine (BR-I2). Molecular Cell 2003 11, 865-874DOI: (10.1016/S1097-2765(03)00096-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 Fitted Gaussian Orientation Distribution Parameters for Each of the BR-Labeled TnCs Relaxed, active, Ca2+-free rigor, and Ca2+-rigor values were measured at 2.4 μm sarcomere length (full overlap). θg and σ are the peak angle and dispersion of the distribution respectively. Model parameters were fitted to data from individual fibers and then averaged. In the case of the C and E helix probes, the relaxed and active values obtained at 2.4 μm sarcomere length are compared with similar data obtained at 4.0 μm sarcomere length (nonoverlap). Data (mean ± SD) were obtained from five to ten fibers for each probe. Molecular Cell 2003 11, 865-874DOI: (10.1016/S1097-2765(03)00096-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 Ribbon Representation of the Lowest Energy NMR Structure of sNTnC.2Ca2+.TnI115-131.BR56-63 The representation is based on Mercier et al. (2003), viewed along the D helix, as in Figure 1C. The BR is shown in stick representation with the Z,Z configuration of the C-N partial double bonds that join the linker arms to the xanthene ring system, as determined by Mercier et al. (2003). Residues 5–85 of TnC and 115–127 of the TnI peptide are displayed; residue 115 is at the end closer to the D helix. Calcium atoms are shown as gray spheres. Molecular Cell 2003 11, 865-874DOI: (10.1016/S1097-2765(03)00096-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Maximum Entropy Distributions for the N Lobe of TnC in Various Physiological States Orientation distributions of the N lobe of TnC with respect to the filament were estimated from measured <P2> and <P4> values (Table 1) by maximum entropy and are shown as contour plots. β is the angle between the D helix and the actin filament axis; γ describes rotation of the N lobe around the D helix. Hotter colors indicate a higher probability of that (β, γ) orientation. The orientation distribution for the N lobe of TnC in (A) relaxed and (B) Ca2+-free rigor muscle using the in vitro APO structure of Gagné et al. (1995). The N lobe distribution in (C) active contraction and (D) Ca2+-rigor are based on the structure of Mercier et al. (2003) (Figure 3). The white crosses shown in (B)–(D) indicate peak β and γ values of the maximum entropy distribution observed in relaxed muscle in (A). Molecular Cell 2003 11, 865-874DOI: (10.1016/S1097-2765(03)00096-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 5 In Situ Orientation of the N Lobe of TnC on the Actin Filament Orientations are derived from peak β and γ values of the maximum entropy distributions shown in Figure 4. The left panel shows the side view of TnC orientation with respect to actin; the right panel shows the same structure, but rotated 90° azimuthally around the actin filament. (A) Orientation of the N lobe of TnC in relaxed muscle based on the Gagné et al. (1995) structure for the APO state. (B) Orientation of the N lobe of TnC in active contraction based on the Mercier et al. (2003) structure for the CA state. Molecular Cell 2003 11, 865-874DOI: (10.1016/S1097-2765(03)00096-0) Copyright © 2003 Cell Press Terms and Conditions