A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy

Transcription

1 Nature Methods A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy Luis A Campos, Jianwei Liu, Xiang Wang, Ravishankar Ramanathan, Douglas S English & Victor Muñoz Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Table 1 Performance of the Trolox-cysteamine mix in proteins labeled with the A488-A594 pair. Cysteamine dose effects on the A488-A594 smfret pair. Comparing the performance of photo-protection strategies for Cy3-Cy5 and A488-A594 FRET pairs. The chemical unfolding of labeled α-spectrin SH3. Comparing the microsecond emission performance of Cy3-Cy5 and A488-A594 in optimal photoprotection conditions.

2 Supplementary figure 1. Performance of the Trolox-cysteamine mix in proteins labeled with the A488-A594 pair. Performance of the Trolox-cysteamine cocktail on proteins labeled with the A488-A594 smfret pair. (a) Number of 100 µs bins with > 25 total photons over background emitted by freely diffusing single molecules per minute as a function of irradiance with a 488 nm CW laser. (Blue) 1 mm Trolox and 10 mm cysteamine, (red) 1 mm Trolox and (green) control experiments without either. The open circles indicate the detected bins that had only donor emission (zero FRET-efficiency peak). The relative performance of the Trolox-cysteamine mix on these dyes was similar for DNA and for proteins labeled on cysteine residues via a thioether bond with maleimide-derivatized dyes. In this case, however, we used lower irradiance than in the experiments with the B-DNA molecule to avoid photo-dissociation of the dyes from the protein. Without any protection we detected a low number of high photon emission bursts. In this case (lower irradiance) the addition of 1 mm Trolox increased the number of high emission 100 µs bursts by 3.5-fold, and reduced the zero-peak down to 30%. However,

3 with the Tx-cyst mix we obtained again the strongest photoprotection, resulting in a 40-fold increase in the number of detected high emission bursts and further reduction of the zero-peak. (b-d) FRET efficiency histograms calculated with 100 µs bins of > 25 photons collected over 5 minutes for the three conditions. The color code is the same as in (a). Without any photo-protection, a large fraction of the events (~70%) corresponded to molecules with only donor emission (zero FRET-efficiency peak). Trolox, and Tx-cyst to a larger extent, decreased the fraction of molecules in the zero FRET-efficiency peak and improved the shape of the FRET-efficiency distribution.

4 Supplementary figure 2. Cysteamine dose effects on the A488-A594 smfret pair. a

5 Effect of cysteamine on the FRET properties of the A448-A594 pair in single protein molecules. (a) Effect on the quantum yield of samples with free A488 (red filled circles), free A594 (blue filled circles), A488-labeled protein (red empty circles) and A594-labeled protein (blue empty circles). Quantum yields were measured in bulk fluorescence experiments on a steady-state fluorometer using Rhodamine 6G and Cresyl violet perchlorate as references. These measurements were made with protein or B-DNA, obtaining very similar results. Cy3 and Cy5 showed little quenching with cysteamine in the mm range, but A488 and A594 did show more marked decreases in fluorescence brightness upon addition of cysteamine. These experiments showed that the quantum yield decrease is approximately linear with cysteamine concentration, but the quenching effect is much stronger for A488 (donor) than for A594 (acceptor). Te decrease in quantum yield was small at concentrations of 10 mm or below, but became quite pronounced as cysteamine concentration increased from 20 to 100 mm. (b) Plot of the total number of 100 µs bursts with > 25 total photons for samples including 1 mm Trolox and different concentrations of cysteamine at different irradiance with a CW 488 nm laser: 0 mm (green), 5 mm (cyan), 10 mm (dark cyan), 15 mm (blue) and 50 mm (dark blue). (c) Plot showing the effect of cysteamine on the high emission properties of A488-A594 labeled protein samples with 1 mm Trolox (light blue) and without (magenta) at an irradiance of 100 kw cm -2. These data were obtained in the same conditions described in Supplementary Fig. 2. (d) Graph showing the fraction of zero FRET-efficiency peak at varying concentrations of cysteamine. Color code as in (b). (e) Dose effect of cysteamine on the fraction of zero FRET-efficiency peak observed for samples with Trolox (dark cyan) and without (magenta) using an irradiance of 100 kw cm -2. Photobleaching kept decreasing linearly (lower population of the zero FRET-efficiency peak) with further increases in cysteamine concentration. However, in that regime the quantum yield decreases became more pronounced so that the overall photon throughput

6 decreased. These results demonstrated that 10 mm cysteamine together with 1 mm Trolox were the optimal conditions for microsecond resolution smfret experiments.

7 Supplementary figure 3. Comparing the performance of photoprotection strategies for Cy3-Cy5 and A488-A594 FRET pairs. a b Detection of high photon-emission bursts (bins of 100 µs with > 40 photons over background) from freely diffusing B-DNA single molecules over a period of 5 minutes at different values of irradiance. (a) Comparison between Cy3-Cy5 (red) and A488-A594 (blue). We observed that the Cy3-Cy5 and A488-A594 pairs have similar FRET properties and produce comparable emission rates in free diffusion smfret experiments without photo-protection. Cy3-Cy5 is a smfret pair that has frequently been used for studies on nucleic acids. These dyes differ from the A488-A594 pair in that they undergo cis-trans isomerization that produces a very long-lasting dark state, which can be reverted by a laser pulse at lower wavelengths 1. This additional photo-blinking mechanism is likely to affect the performance of the various photo-protection strategies. (b) Comparison of photoprotection strategies on single B-DNA molecules labeled with the Cy3-Cy5 pair. (Green) in the presence of oxygen without photoprotection; (magenta) in the presence of 2 mm Trolox plus the enzymatic oxygen scavenger cocktail; (blue) 1 mm Trolox plus 10 mm cysteamine. The lines are polynomial fits to guide the eye. The Tx-cyst mix appears to be much

8 less efficient increasing the photon emission rates of Cy3-Cy5 than of the A488- A594 pair. The number of high emission bursts detected in free diffusion smfret experiments is in general similar with the Tx-cyst mix and without any protection unless the irradiance is increased up to ~1,000 kw cm -2. The enzymatic oxygen scavenger system, on the other hand, showed the opposite behavior. At lower irradiance (for example 100 kw cm -2 ), the oxygen scavenger system is very efficient and increases the number of high emission bursts by almost an order of magnitude. However, at higher irradiance the effect of the enzymatic oxygen scavenger system was detrimental, producing significantly lower number of high emission bursts than without protection. References 1 Heilemann, M., Margeat, E., Kasper, R., Sauer, M. & Tinnefeld, P. Carbocyanine dyes as efficient reversible single-molecule optical switch. JACS 127, (2005).

9 Supplementary figure 4. The chemical unfolding of labeled α-spectrin SH3. a b c (a) We determined the equilibrium chemical denaturation of α-spectrin SH3 in bulk measuring the FRET-efficiency between A488 as donor and A594 as acceptor. The protein underwent a typical sigmoidal unfolding transition upon addition of chemical denaturants, such as guanidinium chloride. Fitting this curve to a two-state transition rendered the parameters: ΔG H2O =13 kj/mol and m eq =2.42 kj/(mol M), which were in excellent agreement with the original parameters from equilibrium and kinetic bulk measurements 1. (b) Comparison of the width of the F and U peaks on the FRET-efficiency histogram of α- spectrin SH3 at 5 M urea obtained with 1 ms bursts with > 200 photons and that expected from shot-noise (green curves). c) Comparison for the FRETefficiency histogram obtained with 100 µs bursts with > 50 photons after eliminating single-molecule trajectories that showed signs of photo-deactivation. Red curves are the expectation from shot-noise. The peaks corresponding to the folded and unfolded states were broader than those in (b) due to the larger contribution from shot-noise, but the overall width is in close agreement with the expectation from shot-noise.

10 References 1 Viguera, A.R., Martinez, J.C., Filimonov, V.V., Mateo, P.L. & Serrano, L. Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-state folding transition. Biochemistry 33, (1994).

11 Supplementary table 1. Comparing the microsecond emission performance of Cy3-Cy5 and A488-A594 in optimal photoprotection conditions. Comparison between the photon throughput (number of 100 µs bursts with > 50 total photons) of B-DNA labeled with Cy3-Cy5 (non-protected and protected with 2mM Trolox plus enzymatic oxygen scavenger) and with A488-A594 (without protection and protected with 1mM Trolox plus 10mM cysteamine). The irradiance was 90 (low) and 300 (high) kw/cm 2 for Cy3-Cy5 using 532 nm excitation, and 110 (low) and 360 (high) kw/cm 2 for A488-A594 using 488 nm excitation.