Fig 1.
ASR1 secondary structure in different environments.
CD spectra of ASR1 (10 μM). A) At different TFE concentrations from 0 (thick line) to 50%, inset: Mean Residual Molar Ellipticity at 222 and % of calculated α-helix as a function of TFE concentration. The solid line corresponds to the fit of 1:1 binding model (Eq 4) B) At different GdnCl concentrations (0 M in thick line); inset, differential spectrum between 6 M and 0 M. C) At increasing temperatures (from 5 to 90°C); inset, differential spectrum between 5°C and 90°C. D) At pH 2, 3, 4, 5, 6, 7 and 8; inset, differential spectrum between pH 5 and pH 8. E) At increasing amounts of NaCl; inset, difference spectrum between 1 M and 0 M. F) Without any stabilizing or denaturant agents (full line) in the presence of crowding agents: 40% of PEG 4000 (dash line) or 50% glycerol (dotted line). More noticeable spectral changes are shown in arrowheads in the inset figures.
Fig 2.
A) CD spectrum changes for ASR1 (2 μM) after the addition of increasing amounts of ZnCl2; inset: Mean Residual Molar Ellipticity at 222 nm and % of calculated α-helix as a function of Zn2+ concentration. The solid line corresponds to the fit of 1:1 binding model (Eq 5). B) Elution profile of ASR1 (63 μg) by SEC using an analytical Superdex 75 column: without Zn2+ (full line) and with 10 μM Zn2+ (dash line); inset: Molar weight determination by SLS for the protein without Zn2+ addition. C) DLS measurements of 40 μM ASR1 with 0, 5 and 10 μM Zn2+. Frequency histogram of hydrodynamic radius (logarithmic scale) is plotted with the corresponding standard deviation.
Fig 3.
Zn2+ effects on ASR1 functions.
A). CD spectrum at 25°C of ASR1 (2 μM) with oligoC (15 μM) full line corresponds to the oligo-protein mixture and dash line represents the algebraic sum of the individual spectrum; inset: spectra of the ASR1 (full line) and oligoC (dash line) alone. B) Fluorescence anisotropy titration. Binding experiments were performed by adding recombinant ASR1 protein to a fixed amount of FITC dsDNA (250 nM). Measurements performed in buffer H with Zn2+ (100 μM): FITC-oligoC (full circles) and FITC-oligoS (full triangles) and without Zn2+: FITC-oligoC (empty circles). Lines correspond to the best fit to the Eq 5 and error bars correspond to the standard errors of two individual experiments. Inset: Fluorescence intensity change as a function of ASR1 concentration performed on nearly saturating conditions (FITC-oligoC: 1250 nM). The arrow indicates the 1:1 stoichiometry. C and D) CS (100 nM) was incubated at 43°C at the indicated ASR1:CS ratios in absence (dashed line) or presence (full line) of 50 μM Zn 2+. +Trehalose indicates the addition of 100 mM trehalose. Plots show light scattering (OD360nm) relative to the highest value obtained on the CS alone sample. CS alone and ASR1 alone were used as controls. Mean values of at least 3 measurements.
Fig 4.
A) Description of full-length FRET reporters. B) Illustration of single tagged-ASR1 constructs. C) Diagram of possible events that may modify FRET efficiency of the ASR1 sensor. D) Fluorescent spectra of purified sticky and non-sticky reporter proteins in the presence or absence of 1 mM Zn2+. E) In vitro Zn2+ titration (1 μM protein) in low micromolar and nearly millimolar ranges (inset) for sticky (circles), non-sticky (triangles) and equal amounts of both single-tagged proteins (stars). Relative (Idonor/IIsosbestic) ratios are shown. Lines correspond to the best fit to Eq 5 for the low micromolar range and Eq 7 for nearly micromolar range. F) NaCl-induced stress assays in E. coli cells expressing sticky (circles) or non-sticky probes (triangles). Relative (Idonor/IIsosbestic) ratios as a function of NaCl concentration are shown.