7 Microfluidic measurements of the substrate protein MBP
7.2 Folding kinetics of DM-MBP
7.2.5 GroEL and GroES mediated refolding of DM-MBP
because of interchanging molecules between the two FRET populations or more likely due to a the low number of photons. The applied flow decreases the time the molecule spends in the focus and, thus, the number of photons is decreased.
In summary, the binding of GroEL to DM-MBP happens on a timescale of approximately 50 ms, which is larger than the diffusing time we calculated of GroEL. After binding, first the low FRET population is filled, which corresponds to unfolded proteins (Figure 7.15). Till approximately 300 ms, the low FRET peak shifts to slightly lower FRET values with a kinetic time of 168 ±59 ms. From 343 ms onwards, the conformation stays stable over time.
Table 7.3: Distances calculated using PDA for DM-MBP with GroEL, GroES and ATP.
DM-MBP (50 pM) was measured in the presence of 0.13 M GuHCl and 3 µM GroEL, 6 µM GroES and 2 mM ATP. The photons of a burst were divided into bins of 1 ms, summed up in a FRET histogram, and fitted by a double-Gaussian model. With photon distribution analysis (PDA), the distance d between donor and acceptor, the corresponding width σ and the relative weights of the different populations were calculated.
Subpopulation Subpopulation Goodness of the fit d [˚A] σ [˚A] % d [˚A] σ [˚A] % χ2
+ GroEL + GroES + ATP 45 3 61.0 48 7 39.0 1.7
To analyze the function of GroEL, GroES and ATP complex on the folding kinetics in more details, microfluidic measurements were performed. Therefore, DM-MBP was denatured in 3 M GuHCl and then diluted to 1.8 M GuHCl in buffer containing 20 mM KCl and 0.001 % Tween20. This solution was then connected to the sample inlet of the microfluidic device.
The buffer inlets were each connected to a buffer solution containing 20 mM KCl, 0.001 % Tween20, 3.25µM GroEL, 6.5 µM GroES and 10.85 mM ATP. This results in a final concen-tration of 3 µM GroEL, 6 µM GroES, 10 mM ATP and 0.13 M GuHCl after mixing.
The FRET efficiency histograms measured at different time points after mixing are depicted in Figure 7.19. After 33 ms, the FRET efficiency histogram is similar to that in the absence of GroEL, GroES and ATP. This can be explained by the fact that binding of GroEL takes longer than 33 ms. Furthermore, it can be seen that already after 50 ms a FRET efficiency of ~0.65 was reached. This FRET value was identified in the measurements without a chap-erone as the intermediate state, which was reached after approximately 500 ms without a chaperone. Thus, GroEL, GroES and ATP speed up the fast collapse to the intermediate state by a factor of 10.
After 232 ms, the FRET efficiency starts shifting to higher values and gets asymmetric. Be-tween 232 ms and 343 ms, the mean FRET efficiency is ~0.75. From there onwards the mean FRET efficiency value is stable at 0.8. The reason for this observation might be that a sec-ond peak at approximately 0.85 is gradually populated. This peak would correspsec-ond to the completely folded DM-MBP. After 1.07 s, the second population is clearly visible.
33 ms
50 ms
155 ms
232 ms
343 ms
512 ms
530 ms
718 ms
792 ms
1.07 s
+ GroEL + GroES + ATP (equilibrium) unfolded
Figure 7.19: Timescale of the refolding kinetics of DM-MBP mediated by GroEL, GroES and ATP. DM-MBP was unfolded in 3 M GuHCl. The GuHCl concentration was diluted to 1.8 M and then mixed in the microfluidic device 1 to 12 with a buffer containing 20 mM KCl, 3.25µM GroEL, 6.5 µM GroES, 10.85 mM ATP and 0.001 % Tween20 (after mixing 3 µM GroEL, 6 µM GroES and 10 mM ATP) to a final concentration of 0.13 M GuHCl. The concentration of DM-MBP was chosen as 15 pM after mixing.
To further analyze the kinetics, the FRET histograms of the different time points after mixing were fitted using Gaussian functions. For the first three measurements, until 155 ms after mixing, only two Gaussian functions were needed to approximate the FRET efficiency his-togram. For the remaining measurements, three Gaussian functions were used. For estimating the mean FRET efficiency values, all FRET efficiency histograms were fitted independently and the mean of the FRET values was determined. The FRET efficiency of the first pop-ulation was determined as 0.47, the second as 0.71, which is similar to the collapsed state, and the last one as 0.87, which corresponds to the folded state. Next, the FRET efficiency histograms were fitted a second time and the mean FRET efficiency value were fixed and the width and the amplitude of the Gaussian were kept free for fitting. The resulting amplitude of the Gaussian functions were plotted versus the time after mixing (Figure 7.20). The indi-vidual populations can be approximated using a linear function. The slope of the curves was determined as−4.48·10−6±2.43·10−6 for the low FRET population,−6.93·10−7±2.44·10−6 for the intermediate FRET population and 3.07·10−6±1.36·10−6 for the high FRET state.
The standard errors were too large to be significant. However, we suggest that the low
FRET population shifts depending on the time after mixing to the folded population and the collapsed population stays on the measured time scale independent.
Amplitude
low FRET (E = 47 %) intermediate FRET (E = 71 %) high FRET (E = 87 %)
Figure 7.20: Kinetic of the GroEL, GroES and ATP mediated refolding of DM-MBP. The FRET efficiency curves of Figure 7.19 were fitted using two or three Gaussian functions. The mean FRET efficiency values were fixed to 0.47, 0.71 and 0.87.
The amplitude of the mean FRET efficiencies of the fit and the corresponding standard error are plotted versus the time after mixing for DM-MBP refolded in 0.15 M GuHCl and 20 mM KCl. The three FRET population were fitted using a linear function resulting in a slope of−4.48·10−6±2.43·10−6,−6.93·10−7± 2.44·10−6 and 3.07·10−6±1.36·10−6, respectively.
Thus, the chaperone system GroEL in combination with its co-chaperone binds between 33 ms and 50 ms and speeds up the folding to a collapsed state by a factor of 10. Furthermore, GroEL-GroES shifts the DM-MBP from an unfolded, i.e. low FRET, conformation to a folded, i.e. more condensed, conformation starting 232 ms after mixing and, after 1.07 s, a fraction of DM-MBP molecules is already, properly folded in its native conformation.