Supplemental Information. Ca 2+ and Myosin Cycle States Work as Allosteric Effectors of Troponin. Activation

Transcription

1 Biophysical Journal, Volume 115 Supplemental Information Ca 2+ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation Christopher Solís, Giho H. Kim, Maria E. Moutsoglou, and John M. Robinson

2 Donor-acceptor pair selection by time correlated single photon counting spectroscopy Probe selection was made by estimating transfer efficiencies of fluorescently labeled regulated actin filaments by time-correlated single photon counting (TCSPC) spectroscopy. Briefly, Tn constructs containing a single-cys TnC (C35S, C84S, T127C), abbreviated TnC-127C, and a single-cys mutants TnI (C81S, C98I, I(X)C), abbreviated TnI-(X)C ((X) represents 151,160, 167,174, 177,182), were fluorescently labeled and reconstituted into regulated actin filaments as described in Materials and Methods. 500 nm of regulated actin with donor only or donor-acceptor in working buffer (75 mm KCl, 50 mm MOPS ph 7.0, 5 mm MgCl 2, 2 mm EGTA, 5 mm 2-mercaptoethanol) at pca 9 or pca 3 estimated using Maxchelator (1). Reconstituted filaments were analyzed using a MicroTime 200 confocal fluorescence lifetime microscope (PicoQuant GmbH, Berlin, Germany) based on an inverted microscope (IX71, Olympus USA, Center Valley, PA). Samples were placed on glass coverslips ( mm width, ThermoFisher Scientific, Pittsburgh, PA) and incubated at room temperature (18 ± 2 ºC) for 30 min prior to measurements. Excitation light from a 532 nm pulsed diode laser (LDH-P-FA-530-B, PicoQuant) was passed through a quarter wave plate, a single mode fiber optic, a laser cleanup filter (534/635-25, Semrock, Lake Forest, IL), a dichroic mirror (DC1) (ZT532/638rpc, Chroma, Bellows Falls, VT), and a 100x (N.A. 1.3) oil immersion objective (UPlanFLN, Olympus). Emitted light was passed through the objective and DC1, then through a 550 nm long pass filter (HQ550lp, Chroma), 50 µm pinhole, a secondary dichroic (ZT532/638PC, Chroma), a bandpass filter (HQ580/70, Chroma), and recorded on an avalanche photodiode (MPD PDM series φ=100 µm, Micro Photon Devices, Italy), respectively. Data were collected until the maximum count per channel exceeded 10,000 (typically 10 min). Background intensity averaged 80 counts/sec. An iterative re-convolution algorithm (SymphoTime Version 5.2, PicoQuant) was used to fit the photon counting histograms of donor emission channel TCSPC with the instrument response function (IRF) using a two- (donor-only) or three- (donor-acceptor) exponential decay model: I(t) = α! e (!!/!!)! (S1), where intensity I(t) is defined by the pre-exponential factor α and the decay time τ. The amplitude-weighed lifetime <τ> was calculated by τ =! α! τ! (S2) for the regulated actin with donor-only (<τ D >) and donor-acceptor (<τ DA >) pairs. The FRET efficiency E was calculated by E = 1!!"!! (S3). The interprobe distance (R) between the donor and acceptor dyes is calculated by the Förster relation: R = R!!!! 1!/! (S4), where f A is the labeling efficiency of the acceptor (here, f A = 0.33) R 0 represents the Förster distance for a particular combination of donor-acceptor probes defined by R! = κ! n!! Q! J λ!/! (S5), where κ 2 is the orientation factor (here, κ 2 = 2/3), n is the refractive index (here, n = 1.33),

3 and Q D is the quantum yield of the donor (for AF 546, Q D = 0.79); J(λ) is the spectral overlap defined by J λ =!!!!!!!!!" (S6),!!!!" where F D (λ) is the emission spectra of the donor (AF 546), and ε A is the extinction coefficient of the acceptor. Using an acceptor probe in TnC127C (ATTO 655) and a donor probe in any of the single-cys TnI positions (Alexa Fluor (AF) 546), the recovered transfer efficiencies from regulated actin filaments containing donor-acceptor constructs show that the largest extent of Ca 2+ -dependent change (ΔE), and interprobe distance decrease (ΔR), is for TnI151-AF 546 found in the switch region (Table S1, Fig. S1); this finding is in agreement with previous work where fluorescent probes were located in TnC89C and TnI151C (2). However, the FRET pair containing fluorescent probes in TnI182 and TnC127C was selected in the present work to TnI mobile domain dynamics. TABLE S1 FRET efficiency and inter-dye distances of acceptor TnC-127C and donor TnI-(X)C pairs in regulated actin. TnI(X)-D position E (%) R (Å) ΔE (%) ΔR (Å) pca 9 pca 3 pca 9 pca ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.0 * Mean ± SD, n = 5. Source, (3). A B C ΔE (%) Switch Mobile domain TnI donor position Normalized intensity D Abs D Em A Abs A Em Overlap R 0 = 59.5 Å Wavelength (nm) TnI donor position FIGURE S1. Selection of donor-acceptor pair position in the Tn construct. (A) Ca 2+ -dependent changes in the transfer efficiencies from donor-acceptor pairs in Table S1 are shown with respect to their position in the TnI switch peptide or the mobile domain (Mean ± SD, n = 5). (B) The absorption (dashed lines) and emission spectra (continuous lines) of the donor (AF 546, green) and acceptor (ATTO 655, red) are used to compute the R 0 distance (59.5 Å) that is a function of the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum (green shade). (C) Ca 2+ -dependent changes in the interprobe distances are computed from the transfer efficiencies in (A) and the estimated R 0 from (B). The pair composed by TnC-127C acceptor and TnI-182C donor shows the larger extent of Ca 2+ -induced interprobe distance change from the probes located in TnI mobile domain (Mean ± SD, n = 5). ΔR (Å) Switch Mobile domain

4 The corresponding donor-acceptor probes were switched to donor-tnc and acceptor-tni because it was found that the acceptor probe is labeled more efficiently on TnI and because the TnC appears to be released from the Tn complex more frequently. This approach is intended to maximize the probability of donor-acceptor pairs in Tn by assigning the donor probe to Tn. To discard any Tn complex dissociation during our experimental conditions, we performed a time-dependent colocalization analysis of regulated actin filaments labeled with donor-acceptor probes to determine the construct stability under diluted conditions (20 nm). Our results show that regulated actin filaments are stable under these conditions and no significant dissociation is expected to occur during the FLIM acquisition time. (Fig. S2). 0 min 10 min 20 min 40 min 60 min FIGURE S2 Regulated actin filaments labeled with donor-acceptor probes are stable under diluted conditions. Regulated actin filaments reconstituted with Tn-donor-acceptor (TnI182C- ATTO655, TnC127C-AF 546, TnT), Tm and F-actin were surface deposited on coverslips at low concentration (20 nm) in the absence of myosin as described in the Materials and Methods section. Merged images display donor (green) and acceptor (red) fluorescence. In vitro motility assay To determine the functional effects of the fluorescent probes covalently bound to Tn, we developed an in vitro motility assay. The in vitro motility assay setup is similar to the procedure described in Materials and Methods. The following modifications were included. (i) Prior to reconstituting regulated actin, 7 µm of F-actin were labeled with 0.35 µm of phalloidin AF488 (Life Technologies) for 20 min before regulated actin reconstitution. (ii) After deposition of regulated actin, or regulated actin labeled with donor-acceptor probes (DA: TnC127C-AF 546, TnI182C-ATTO 655), the in vitro motility assay was initiated by addition of imaging buffer supplemented with 5 mm ATP, 0.1 µm Tm, and 0.1 µm Tn or 0.1 µm Tn-DA. In the case of Tn-DA measurements, it was necessary to briefly photobleach the AF546 signal to minimize spectral bleedthrough into the phalloidin AF488 signal. Wide field epifluorescence images were acquired on an inverted IX71 Olympus microscope with cooled interline CCD camera (Clara, Andor) using a 100x (N.A. 1.4) oil immersion objective (UPlanSApo, Olympus). Videos were acquired at 5 frames per second in a 512x512 pixel field of view. Excitation was from a mercury vapor lamp (XCite 120PC, Lumen Dynamics). Filters (excitation: dichroic: emission) were FF01475/35: FF495: FF01-550/88 (Semrock) for AF 488. The CCD camera field of view was calibrated by imaging a dual axis linear scale (Edmund Industrial Optics). Videos were analyzed using a previously published filament tracking algorithm (4).

5 In vitro motility assays show Ca 2+ -dependent activation of regulated actin and regulated actin with DA sliding without significant differences at pca 3 and pca 9 (Fig S3, Video S1). These results indicate that covalent crosslinking of fluorescent probes in Tn does not interfere with functional properties of reconstituted filaments. A pca 3 pca 9 B Speed (µm/s) FIGURE S3 FRET-pair labeling of Tn does not inhibit filament and Ca 2+ activation. (A) Representative frames of regulated actin filaments sliding at pca 3 and pca 9 depicting motile (green) and static filaments (red) (scale bar, 2 µm). (B) Average filament sliding speeds at pca 3 (gray) and pca9 (white) of regulated actin with WT Tn (Control) and Tn labeled with donor-acceptor probes (mean ± SD; n = filaments per treatment, derived from three separate experiments; T = 18 ± 2 ºC). FLIM-FRET data analysis WT Donor-Acceptor To derive quantitative information from our fluorescence lifetime images, we developed an image-processing algorithm to exclude background pixels from the analysis. An image processing routine used to select pixels representing the fluorescence lifetime image from regulated actin filaments (Fig. S4 A). The fluorescence lifetime acquired from the binary mask reduces the background fluorescence (Fig. S4 B).

6 A B Normalized counts (AU) Lifetime (ns) FIGURE S4 Filament recognition in fast-flim. Fast-FLIM images composed by a lifetime and an intensity channel were analyzed using a customized image-processing algorithm. (A) A fast-flim image of regulated actin-da (left) and after binarization (right) for filament identification. The binary map is used to interrogate the lifetime channel in a fast-flim image (scale bar, 5 µm). (B) The recovered lifetime histograms from the filament lifetime (red) and the entire image (black) have lifetime values of 3.23 ns and 4.97 ns respectively. To provide evidence of statistical difference on Ca 2+ -induced activation, the pca 9 and pca 3 conditions were pooled into two groups. A one-way ANOVA test revealed the pca 9 and pca 3 groups are statistically different (p = 2.03x10-17 ). Based on these results, the differences between the each treatment (Control, ATP-γ-S, ADP-Pi-Bleb, Rigor) were determined by analyzing pca 9 and pca 3 conditions in separate groups. For pca 9 conditions, a pairwise post hoc analysis of the transfer efficiencies based on Tukey s honest significance test revealed significant activation of rigor myosin compared to all the other conditions (p < 0.01); ADP-Pi-Bleb is significantly different (p < 0.01) from control and ATP-γ-S (Table S2). For pca 3 conditions only the rigor state is significantly different from all the other conditions (Table S3). The full width at half-maximum (FWHM) data revealed that only the rigor state is different from control measurements at both pca 9 (Table S4) and pca 3 (Table S5). Pooling the pca 9 and pca 3 FWHM data in a one-way ANOVA analysis shows that pca 9 rigor is significantly different from the pca 3 rigor (p = ); however, the pca 9 control is not significantly different from the pca 9 control.

7 TABLE S2 Pairwise post hoc analysis of FRET efficiencies at pca 9 Condition 1. Control 2. ATP-γ-S 3. ADP-Pi-Bebbistatin 4. Rigor 1. Control 2. ATP-γ-S ADP-Pi-Beb 0.009** 0.008** 4. Rigor 0.000*** 0.000*** ** Significance tests based on Tukey s honest significance test. *p < 0.05, ** p < 0.01, *** p< TABLE S3 Pairwise post hoc analysis of FRET efficiencies at pca 3 Condition 1. Control 2. ATP-γ-S 3. ADP-Pi-Bebbistatin 4. Rigor 1. Control 2. ATP-γ-S ADP-Pi-Beb Rigor 0.000*** 0.000*** 0.005** Significance tests based on Tukey s honest significance test. * p < 0.05, ** p < 0.01, *** p < TABLE S4 Pairwise post hoc analysis of calculated FWHMs at pca 9 Condition 1. Control 2. ATP-γ-S 3. ADP-Pi-Bebbistatin 4. Rigor 1. Control 2. ATP-γ-S ADP-Pi-Beb Rigor 0.005** Significance tests based on Tukey s honest significance test. *p < 0.05, ** p < 0.01, *** p < TABLE S5 Pairwise post hoc analysis of calculated FWHMs at pca 3 Condition 1. Control 2. ATP-γ-S 3. ADP-Pi-Bebbistatin 4. Rigor 1. Control 2. ATP-γ-S ADP-Pi-Beb Rigor 0.004** * Significance tests based on Tukey s honest significance test. *p < 0.05, ** p < 0.01, *** p < Correlation data in Fig. 4 was fitted to a Gaussian mixture model with k number of bivariate Gaussian distributions: P(t,w) = n k= t exp k ˆt k t 2 2 2ρ k ˆt k 2πσ t,k σ w,k 1 ρ 2 1 ρ k ( k ) σ k w ŵ k k + w ŵ 2 k k t,k σ t,k σ w,k σ w,k (S1), where t corresponds to the FRET efficiency E, w to the lifetime histogram FWHM, ˆt k and ŵ k are the mean values of t and w respectively, σ t,k and σ w,k are the standard deviations of t and w respectively, and ρ k is the correlation between t and w. Model selection is based on the Akaike information criterion (AIC) which ranks each model composed by k components according to their relative goodness of fit and simplicity of the model. AIC with a correction applied to small sample sizes (AICc) is defined as: AICc = 2k 2ln( ˆL )+ 2k2 +2k n k 1 (S2),

8 where ˆL is the maximum value of the likelihood estimate and n is the sample size. The model k that minimizes AICc is the model with the relatively highest quality. For our particular case, two models were tested to describe the relation between the FRET efficiency E and lifetime histogram FWHM with k = [1,2]. The optimal model is found to be k = 2. To establish significance between the FRET and the FWHM dependence (i.e. differences in the slopes), an analysis of covariance (ANCOVA) was conducted from the data reported in Fig. 4. Results show that this dependence is not significant (Fig. S5). FIGURE S5 Analysis of the FRET and the FWHM dependency. Analysis of covariance (ANCOVA) of Fig. 4 indicates that the FRET and the FWHM dependence when comparing the pca9 (open circles) and pca 3 (closed circles) groups is not significant (p = 0.07). Red lines denote linear fits for each group and the blue shades correspond to the 95% confidence bounds of each line. References 1. Patton, C., S. Thompson, and D. Epel Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium 35: Robinson, J. M., W. J. Dong, J. Xing, and H. C. Cheung Switching of troponin I: Ca(2+) and myosin-induced activation of heart muscle. J Mol Biol 340: Moutsoglou, M. E A FRET investigation into molecular mechanisms of cardiac troponin activation in reconstituted thin filaments. In Chemistry & Biochemistry. South Dakota State University, Brookings, South Dakota Ruhnow, F., D. Zwicker, and S. Diez Tracking single particles and elongated filaments with nanometer precision. Biophys J 100: