Myofilament Calcium Sensitivity: Consequences of the Effective Concentration of Troponin I

Figures

• 

  Figure 1. Comparison of apparent Ca2+ sensitivities and dissociation kinetics of different states of TnC. Panel (A) shows the apparent Ca2+ sensitivities ± SEM for TnC in the different biochemical systems that were extracted from our earlier publications (Tikunova and Davis, 2004; Davis et al., 2007; Little et al., 2012a,b). Panel (B) shows the apparent Ca2+ dissociation kinetics ± SEM for TnC in the different biochemical systems that were extracted from the same earlier publications. The y-axis is in a log scale making it difficult to see some of the error bars.

• 

  Figure 2. Biochemical testing of the effective concentration of TnI. Panel (A) shows the apparent Ca2+ sensitivities of isolated TnCF27W in the absence or presence of increasing concentrations of TnI128−180 compared to that of the F27W chimera. Panel (B) shows the apparent Ca2+ dissociation rates from isolated TnC T53C-IAANS in the presence of increasing concentrations of TnI128−180 compared to that of the T53C-IAANS chimera. Panel (C) shows the apparent rate of Ca2+ dissociation from T53C-IAANS chimera. Panel (D) shows a representative SDS Page gel of: lane (1) molecular weight markers; lane (2) TEV; lane (3) uncut F27W chimera; lane (4) TEV digested T53C-IAANS chimera; lane (5) TEV digested F27W chimera. The arrows point to the following proteins in descending order TEV, uncut F27W chimera, TnI fragment, TnC T53C-IAANS fragment and TnC F27W fragment, respectively. The TnI fragments from the cut chimeras are of identical sequence and size, suggesting the lowest fragments are TnC. Panel (E) shows the apparent rate of Ca2+ dissociation from the TEV protease digested T53C-IAANS chimera before and after the addition of 30-fold excess TnI128−180.

• 

  Figure 3. Cartoon illustration of the effective concentration of TnI. Panel (A) shows the crystal structure of the Tn complex [cyan TnI (purple switch peptide); red TnC; and yellow TnT]. The blue shaded area represents a hypothetical volume in which the TnI switch peptide may be found. Panel (B) illustrates the potential structural change that occurs in TnI upon Ca2+ removal from TnC and TnI binding to actin.

• 

  Figure 4. Application of a two state model. Panel (A) shows a reaction schematic for a two state model of TnC's Ca2+ binding. Panels (B–G) in black show the apparent Ca2+ sensitivities or dissociation rates from previously published data (Davis et al., 2007; Liu et al., 2012b), overlaid in red with a two-state model simulation. All measurements were made following the change in fluorescence of TnC T53C-IAANS in increasingly complex biochemical systems.

• 

  Figure 5. Role of competitive Mg2+ binding in regulating TnC's apparent Ca2+ sensitivity. Panel (A) shows how implicitly (Ignore Mg2+ Competition) or explicitly (Explicit Mg2+ Competition) affects the “intrinsic” Ca2+ association rates for TnC in different biochemical systems. Panel (B) shows a reaction schematic for a three state model that takes into account explicit Mg2+ binding to TnC. Panel (C) shows the apparent shift in Ca2+ sensitivity of the F27W chimera in the presence (solid squares) or absence (open squares) of 3 mM Mg2+.

• 

  Figure 6. Application of a three state model. Panel (A) shows a reaction schematic for a three state model for TnC activation. Panels (B,C) show plots of the apparent Ca2+ sensitivity and dissociation kinetics derived from a three state model with increasing effective TnI concentration. Panel (D) shows the reaction schematic for a four state model for TnC activation. Panels (E,F) show plots of the apparent Ca2+ sensitivity and dissociation kinetics derived from a four state extended model with increasing effective TnI concentration.

• 

  Figure 7. Application of a four state model. Panels (A,B) in black show the apparent Ca2+ sensitivity and dissociation rates from the Tn complex as previously published (Davis et al., 2007). Panels (C,D) in black show the apparent Ca2+ sensitivity and dissociation rates from the reconstituted thin filament as previously published (Davis et al., 2007). Panel (E) in black shows the rates of Ca2+ association to the reconstituted thin filaments as previously published (Liu et al., 2012b). Panel (F) (noisy colored curves) shows the response of the thin filaments to artificial Ca2+ transients as previously published (Liu et al., 2012b). For Panel (A–E), the smooth red curves show the simulated output of the four state model. For Panel (F), all the smooth curves represent the simulated output of the four state model.

• 

  Figure 8. Changes in Only the Effective Concentration of TnI Predict Ca2+ Binding Properties of Several TnI and TnT Modifications. Panel (A) shows that the apparent Ca2+ sensitivities of several TnI and TnT modifications fall on the four state TnC activation curve as the effective concentration of TnI is increased. Panel (B) shows that the apparent Ca2+ dissociation rates of several TnI and TnT modifications fall on the four state TnC activation curve as the effective concentration of TnI is increased. The exact same effective TnI concentration was used for each specific protein modification in each panel.

• 

  Figure 9. Using the four state model to reconcile data from different research groups and predicting the molecular mechanism for S150 phosphorylation. Panels (A,B) show the apparent Ca2+ dissociation rates and sensitivities for thin filaments containing control or S23/24D TnI, whereas panels (C,D) show the apparent Ca2+ dissociation rates and sensitivities for the Tn complex containing control, S23/24D TnI or S150D TnI. For panels (A–D), the black traces are real data previously published from the Davis lab (Liu et al., 2014), the blue traces are real data previously published from the Biesiadecki lab (Nixon et al., 2014), the red traces are simulations for the Davis lab conditions, whereas the magenta traces are simulations for the Biesiadecki lab conditions.