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2.
Scheme 2

Scheme 2. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

M200 filament (Mn) dissociation by S100A4.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
3.
Scheme 1

Scheme 1. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Ca2+ and myosin peptide (M) binding to S100A4.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
4.
Fig. 7

Fig. 7. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Chemical shift changes in the 1H,15N HSQC spectra of 13C,15N-labelled M111 on addition of full-length S100A4. Superposition of the spectra in free form (black) and in the presence of a 1.5 molar excess of S100A4 (red). Resonance assignments derived from the analysis of triple-resonance spectra are marked. In addition to sharp intense cross-peaks, the spectra contain broad low-intensity peaks that cannot be assigned due to low sensitivity.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
5.
Fig. 2

Fig. 2. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

ITC records for Ca2+ binding to S100A4 constructs: (a) 75 μM wild type at 30 °C; (b) 75 μM wild type at 10 °C; (c) 75 μM E33Q construct at 30 °C; (d) 300 μM D63N construct at 30 °C. For mutant constructs data at 10 °C, see Fig. S1. The fitted lines in the bottom panels correspond to the data shown in Table 1. All preparations were dialysed against the same buffer of 20 mM NaCl, 10 mM Hepes, and 2 mM DTT (pH 7.5).

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
6.
Fig. 6

Fig. 6. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Chemical shift changes in the 600-MHz 1H,15N HSQC spectra of 0.2 mM 15N-labelled Δ10-S100A4 on addition of myosin peptides at 35 °C. (a) Superposition of the spectra in free form (black) and in the presence of a 3-fold molar excess of M32 (red). The inset shows progressive changes in the spectra on peptide addition (intermediate exchange) corresponding to the following protein/peptide molar ratios (left to right): free (black), 1:0.25 (green), 1:0.5 (light blue), 1:1.5 (dark blue), and 1:3 (red). Cross-peaks corresponding to 1:0.75 and 1:1 molar ratios are too broad to be observed. (b) Superposition of the spectra in free form (black) and in the presence of a 4-fold molar excess of M16N (red). The inset shows progressive changes in the spectra on peptide addition (fast exchange) corresponding to the following protein/peptide molar ratios (left to right): free (black), 1:1 (magenta), 1:2 (light blue), and 1:4 1:3 (red). (c) Superposition of the spectra in free form (black) and in the presence of a 4-fold molar excess of M15C (red). The inset shows progressive changes in the spectra on peptide addition (fast exchange) corresponding to the following protein/peptide molar ratios (left to right): free (black), 1:1 (magenta), 1:2 (light blue), and 1:4 1:3 (red).

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
7.
Fig. 8

Fig. 8. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Characterisation of myosin IIA coiled-coil tail fragments. Metal-shadowed electron micrographs of (a) M200 and (b) M111 deposited from 0.5 M ammonium acetate. Scale bar represents 50 nm. (c and d) Electron micrographs of a negatively stained M200 tail fragment. Fields of M200 (c) in the absence and (d) in the presence of a 2-fold molar excess of S100A4, respectively. Arrows point to individual strands emerging from images of filamentous bundles. The samples were prepared in 0.1 M sodium acetate buffer, where M200 formed large filamentous aggregates but were solubilised in the presence of S100A4. Scale bar represents 200 nm (see Materials and Methods for details).

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
8.
Fig. 1

Fig. 1. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Sequence of C-terminal human NM-MHC IIA fragments and peptides. The His-tagged expressed fragments M200 and M111 contained an N-terminal fusion comprising residues MRGSHHHHHGS. The sequence shown corresponds to M200 (residues Q1761-E1960; native C-terminus). M111 starts at residue Q1850 (red). The M32 peptide (A1907-G1938) is underlined, M16N (D1908-R1923) is shown in green, and M15C (G1924-G1938) is shown in blue. Residues in the a and d heptad repeat positions of the coiled coil (generally hydrophobic) are shown in boldface. The nonhelical C-terminus region is shown in italics. Note that the numbering system is one residue shorter than that originally used by Kriajevska et al. and corresponds to the current nonmuscle myosin IIA database nomenclature (NCBI reference sequence NM_002473.4). 9 Thus, M200 is equivalent to the Hmyo4-3B fragment. 9 Note that the M200 and M111 preparations contain a V1913I substitution.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
9.
Fig. 10

Fig. 10. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

(a) Simulated response of the kinetic pathway (Scheme 1) to a single wave of Ca2+ (broken line) with a rise time constant of 10 ms and a decay constant of 100 ms. For simplicity, the myosin filament was modelled as a cooperative system 5M ↔ M5 (see Fig. S7 for further details). The total concentrations of the components were 2 μM S100A4 and 2 μM myosin (monomer concentration). Filament (black line), S100A4–Ca2+ (red line), and M–S100A4–Ca2+ (blue line). (b) As in (a), but with the response to a step increase in free Ca2+ to 2 μM. (c) FRAP of GFP–myosin IIA expressed in an A431/SIP1 cell immobilised on a Y-fibronectin pattern (CYTOO Chip™). The TIRF image is derived from a sequence 1 s after the bleaching of a 3-μm-diameter spot (broken circle) across a stress fibre. Scale bar represents 10 μm. (d) FRAP time course of the bleached spot in (c), normalised against an unbleached region of the filament, to yield a recovery rate constant of 0.046 s− 1.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
10.
Fig. 3

Fig. 3. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

(a) Dissociation of Ca2+ from wild-type S100A4 (black), E33Q mutant (blue), and D63N mutant (red), as monitored by Quin-2 fluorescence. Fits to a single exponential yielded rate constants of 16 s− 1, 35 s− 1, and 500 s− 1, respectively, but the D63N record had a small amplitude, suggesting that most of the signal change was too fast to measure. The records for the mutants were vertically offset for clarity. (b) Ca2+ binding to S100A4 as determined from the amplitude of the resolved transient (k ≈ 20 s− 1) when 6.5 μM S100A4 was preincubated with various added [Ca2+] and then mixed with 100 μM Quin-2. The amplitude was converted into a bound concentration from an independent calibration using the same Quin-2 solution and known free [Ca2+]. The latter transients were too fast to measure but gave a steady signal on the seconds timescale. The continuous black line was computed for a model with cooperative binding in which the first Ca2+ bound to EF2 of an S100A4 dimer with a Kd of 100 μM and the second Ca2+ bound to EF2 of an S100A4 dimer with a Kd of 1.7 μM. The blue line was computed for a model with an EF1 binding site with Kd = 0.25 μM that was optically silent in the Quin-2 assay (i.e., dissociation too fast to measure) and an EF2 site with Kd = 5 μM. While either model could account for the data, the measured stoichiometry from ITC (Table 1) favors the cooperative model.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
11.
Fig. 5

Fig. 5. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Dissociation of Ca2+ from S100A4 in the absence and in the presence of target peptides monitored using Quin-2 fluorescence. (a) Mixed with 100 μM Quin-2 was 10 μM S100A4 + 50 μM Ca2+ (stopped-flow reaction chamber concentrations). A superposed fit to a single exponential yields kobs = 17 s− 1. (b) As in (a), but with 50 μM M16N premixed with S100A4 (kobs = 16 s− 1). (c) As in (a), but with 50 μM M32 premixed with S100A4 (kobs = 3.3 s− 1). (d) As in (a), but with 2.5 μM M200 premixed with 5 μM S100A4. A biphasic exponential fit yielded rate constants of 13 s− 1 (A = 26%) and 0.30 s− 1 (A = 74%). The broken line shows increased light scatter at 336 nm as the released M200 formed filaments in the absence of Ca2+ with a 10-s lag and indicates that this process did not influence the profile of Quin-2 fluorescence. Experiments were carried out in either 20 mM NaCl, 20 mM Tris, 1 mM DTT, and 100 μM Ca2+ at pH 7.5 and 20 °C (a and b; cf. Malashkevich et al. 17 ), or 100 mM NaCl, 10 mM Hepes, and 100 μM Ca2+ at pH 7.5 and 20 °C (c and d). Similar profiles were observed for experiments (a and b) performed in the latter buffer and yielded rate constants of 16 s− 1, indicating a weak binding of M16N under both conditions. Rate constants were unaffected by the inclusion of 1 mM Mg2+.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
12.
Fig. 9

Fig. 9. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

Dissociation of M200 filaments by S100A4. (a) Initially, M200 was added (i) from a concentrated stock in 0.5 M NaCl to give a final concentration of 5 μM M200 (with respect to the polypeptide chain) in 100 mM NaCl, 10 mM Hepes, and 0.1 mM Ca2+ at pH 7.5 and 20 °C. A biphasic rise in turbidity was recorded for 200 s (continuous line). Then 10 μM S100A4 (with respect to monomer concentration) was added (ii), followed by 0.2 mM EGTA (iii) and 1 mM Ca2+ (iv). The experiment was repeated in the presence of 250 μM M32 (broken line) or 250 μM M16N (dotted line). (b) M200 was diluted (i) into 100 mM NaCl to form filaments as in (a), then 5 μM S100A4 was added (ii), causing a 54% drop in turbidity. A further 5 μM S100A4 (iii) caused turbidity to fall to almost zero. (c) Stopped-flow records monitoring the dissociation of 5 μM M200 filaments on jumping [NaCl] from 100 mM to 120 mM NaCl. Other components of the buffer were 20 mM Hepes, 1 mM Mg2+, and 100 μM Ca2+ at pH 7.5 and 20 °C. The upper trace, obtained in the absence of S100A4, showed a 45% decrease in turbidity with rate constants of 0.33 s− 1 (22% amplitude) and 0.037 s− 1 (78% amplitude). In the presence of 20 μM S100A4 (lower trace), the turbidity decreased by 87% with a rate constant of 0.2 s− 1. The broken line shows the calculated profile when the disassembly of the M200 filaments occurred only by sequestration of monomeric M200 by S100A4 with a rate constant of 0.04 s− 1, which gives the same initial rate as observed in the absence of S100A4 (see the text).

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.
13.
Fig. 4

Fig. 4. From: Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA.

(a) The binding of fluorescein-labelled M16N myosin peptide (0.1 μM) to S100A4 based on fluorescence anisotropy. Titration (circles) was carried out in 20 mM NaCl, 10 mM Hepes, 1 mM Mg2+, and 100 μM Ca2+ at pH 7.5 and 20 °C. The fitted hyperbola yielded an apparent Kd of 2.0 ± 0.23 μM (see the text for correction). At the end of the titration, EGTA was added to 1 mM, causing the peptide to dissociate (square datum). (b) Displacement of F-M16N (5 μM) from S100A4 (5 μM) by increasing [M32] (circles) or [M16N] (squares). The continuous line corresponds to a Kd of 3 μM for M32 binding to S100A4, as determined by a fit to a competitive single binding site equation, using the Excel solver routine to minimise the variance. The continuous line deviated systematically from the data when the modelled Kd for M32 was increased or reduced by 50% of its value. M16N caused little displacement of its fluorescent counterpart even at a concentration of 200 μM. Modelling to a competitive binding equation indicates that the Kd of M16N for S100A4 is ≥ 100 μM (broken line). (c) Stopped-flow record of 0.1 μM F-M16N binding to 25 μM S100A4 monitored by fluorescein emission intensity. A fit to a single exponential yields kobs = 138 s− 1. (d) Dependence of the pseudo first-order rate constant kobs for F-M16N (0.1 μM) binding as a function of S100A4 concentration. The rate constant of 7.7 s− 1 (open square) at zero [S100A4] was obtained by the displacement of FM16N from S100A4 by excess M32 (see Fig. S2a). The fitted straight line yields kon = 4.9 μM− 1 s− 1 and koff = 6.2 s− 1. At concentrations above 25 μM S100A4, the graph became nonlinear and reached a maximum kobs of 150 s− 1.

Sandip K. Badyal, et al. J Mol Biol. 2011 January 28;405(4-3):1004-1026.

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