Visualizing Temperature Mediated Activation of Gelsolin and Its Deactivation By Pip2: A Saxs Based Study

Figures

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  Figure 1. F-actin depolymerizing activities of gelsolin (at 0.83 μg/ml) are shown here by measuring relative decrement of pyrene fluorescence values as a function of temperature at which gelsolin in different buffers (mentioned in each panel) were heated to before performing the assays.

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  Figure 2. (A) The SAXS I(Q) profiles of gelsolin (8 mg/ml) at different temperatures in buffer lacking any free Ca2+ and pH 8 are plotted here. (B) This panel shows the Kratky plots of the SAXS datasets shown in panel (A). The smoothened versions were generated by averaging adjacent 5 data points). (C) The Guinier analysis of all the datasets presuming globular scattering profiles are shown here. (D) Variation in the molecular mass of the gelsolin molecules estimated using I0 values from Guinier analysis as a function of temperature are plotted here. The blue line represents the Sigmoidal fit to the data presuming two-state transition. Inset shows the image of the native PAGE of the heated samples. Red circles indicate samples where slight aggregation was visible and thus indicate uncertainty in estimation. (E) Variation in the RG values as a function of temperature for gelsolin solution is plotted here (black squares). The blue line fitted through symbols shows the Sigmoidal fit to the observed trend in the increase of RG values. The point of half-change for RG values in samples varying in gelsolin concentration are shown in inset. (F) Variation in the calculated RG values of the…

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  Figure 3. The variations in the mean residue ellipticity (MRE) values at 222 nm in variable temperature CD experiments are plotted here (blue and black squares represent data from gelsolin samples, 0.5 and 1 mg/ml, respectively). The gray shaded zone indicates samples at which whitish aggregation was visible. (A) Buffer having 1 mM free Ca2+ ions and pH of 8, (B) buffer lacking any free Ca2+ ions and pH 5, and (C) buffer lacking any free Ca2+ ions, and pH 8.

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  Figure 4. (A) The P(r) profiles computed using SAXS data profiles collected for 8 mg/ml gelsolin shown in Fig. 2A are plotted here for temperatures 10, 30 and 40 °C. (B–D) The SAXS data based dummy residue model solved for gelsolin protein at temperatures 10, 30 and 40 °C are presented. The dummy residues are shown as grey mesh. As discussed in main text, for each SAXS data based model, either crystal structure of Ca2+-free gelsolin or its simplistic reoriented model was used to assess the shape rearrangements seen in the SAXS data based dummy atom models. In all structural models used with SAXS data based models, the G1 domain and g1–g2 linker, and the C-tail latch are depicted in red and magenta coloured ribbons, respectively. Additionally, the black circle drawn highlights the location of G1 domain and the arrows between different panels track its repositioning as a function of temperature.

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  Figure 5. (A) The variation in the SAXS data based estimated RG values of ΔCT and G2–G6 gelsolin are shown as a function of time relative to the values computed for full-length gelsolin. (B) Variation in the molecular masses of the ΔCT and G2–G6 gelsolin estimated using I0 values from Guinier analysis are compared with those estimated for full-length gelsolin. (C) SAXS data based dummy residue models of ΔCT gelsolin at 10 and 35 °C (red mesh). Crystal structures are placed inside as described in main text. (D) SAXS data based dummy residue models of predominant scattering shape of G2–G6 gelsolin in Ca2+ free buffer having pH 8 at 10 and 45 °C (blue mesh). The right panels in both (C,D) segments show the variation in the RG values of the conformations assessed by EOM calculations.

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  Figure 6. (A) Variation in RG values of gelsolin molecules in absence of PIP2 as tracked by Guinier analyses of the SAXS datasets obtained at different temperatures are shown. The data here shows whether the RG values decrease upon lowering the sample temperature or not. (B) Computed RG values from SAXS datasets obtained from samples of gelsolin mixed with PIP2 are presented here as function of temperature. The blue arrow highlights the PIP2 addition induced decrement in RG values in gelsolin samples pre-heated to 45 °C. (C) SAXS data based scattering shape solved for gelsolin molecules at 40 and 45 °C but without and with PIP2 in solution, respectively are shown here (dummy residues shown as brown mesh). As detailed in discussion section, models were placed inside SAXS data based shape profiles for comparison. (D) The panels show the variation in RG values of gelsolin value as a function of free Ca2+ ions (left) or low pH in buffer (black open squares). The filled red squares show the RG values of the samples having same amount of Ca2+ ion or pH in buffer, plus PIP2. The black arrows highlight decrement in the RG values upon addition of PIP2.

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  Figure 7. Schematic summary of partially and fully open shapes of gelsolin induced by different physiological regulators of its activity i.e. Ca2+ levels, pH and temperature inside and outside cellular environment. Compilation also sums up how PIP2 can revert activation, but only from partially open or activated state.