Spontaneous refolding of DM-MBP

To monitor the conformational changes of the N-terminal domain of DM-MBP upon refolding, microfluidic measurements were performed. As a reference for the unfolded and properly folded DM-MBP, equilibrium measurements were performed. The DM-MBP was denatured in 3 M GuHCl, a chaotropic agent, and, afterwards, the GuHCl was diluted to 2 M. The spFRET (see Chapter 2.5 on page 11) was determined on a multiparameter fluorescence detection setup (MFD, see Chapter 3.3.2 on page 25) with pulsed interleaved excitation (PIE, see Chapter 3.3.1 on page 24). The distance between the two dyes was calculated by photon distribution analysis (PDA, see Chapter 3.3.3 on page 32).

Unfolded in 3 M GuHCl - Diluted in 1.8 M GuHCl Unfolded - 2 M GuHCl

Unfolded in 3 M GuHCl - Diluted in 1.5 M GuHCl Unfolded in 3 M GuHCl - Diluted in 0.2 M GuHCl Unfolded in 3 M GuHCl - Diluted in 0.1 M GuHCl Native

Figure 7.11: DM-MBP measured in different GuHCl concentrations in equilibrium. DM-MBP was denatured in 2 M GuHCl (black line). After denaturation in 3 M GuHCl, 50 pM DM-MBP was measured in 1.8 M GuHCl (dark yellow line), in 1.5 M GuHCl (red line), in 0.2 M GuHCl (cyan line) and in 0.1 M GuHCl (dark blue line). As a reference native DM-MBP was measured (gray line).

The FRET efficiency for the unfolded DM-MBP is approximately 0.1 (Figure 7.11, black line), which corresponds to a distance between the dyes ofd= 75 ˚A and a width ofσ = 13 ˚A (Table 7.1). This width of the distribution is very broad, which can be explained by multiple subconformations due to the flexibility of the unfolded protein.

By reducing the GuHCl concentration to 0.1 M, the DM-MBP folds properly and the FRET efficiency changes to approximately 0.9 (Figure 7.11, blue line). A GuHCl concentration of 0.2 M emerged to be too high to reach the final, proper folded conformation (Figure 7.11, gray line). The FRET efficiency of the native state corresponds to a distance of d= 45 ˚A with a

width ofσ = 4 ˚A (Table 7.1). This width is well defined and, therefore, represents to a single conformation. These results are in line with recently published data [Sharma et al., 2008].

Table 7.1: Distances calculated using PDA for DM-MBP. DM-MBP (50 pM) was measured in the presence of 2 M GuHCl (unfolded) and in the presence of 0.1 M GuHCl (folded). The photons of a burst were divided into bins of 1 ms, summed up in a FRET histogram, and fitted using a Gaussian model. With photon distribution analysis (PDA), the distance d between donor and acceptor, the corresponding widthσ and the relative weights of the different populations were calculated.

Subpopulation Goodness of the fit d [˚A] σ [˚A] % χ²

unfolded 75 13 100 1.3

folded 45 4 100 4.2

The timescale of interchanging between the unfolded and the refolded conformation was monitored by a combination of spFRET and microfluidics. The DM-MBP was denatured again in 3 M GuHCl and, afterwards, diluted to 2 M GuHCl in 5 mM Tris (pH 7.8), 20 mM KCl and 5 mM Mg[OAc]₂. Then, the DM-MBP was connected to the sample inlet of the microfluidic device. The buffer inlets were connected to 5 mM Tris (pH 7.8), 20 mM KCl and 5 mM Mg[OAc]₂. Additionally, 0.001 % Tween20 was added to all solutions to avoid sticking of the protein to the tubes or the microfluidic device. The sample and buffer solutions were mixed in the device in a 1 to 12 ratio, e.g. 1 part DM-MBP and 12 parts buffer, to a final GuHCl concentration of 0.15 M. With the 1 to 12 ratio, two flow rate combinations of the sample and buffer channels can be measured, i.e. 0.2 _min^µl and 0.3 _min^µl applied to the sample channel and 1.2 _min^µl and 1.8 _min^µl applied to each of the buffer channels, respectively. This was the highest reachable dilution in the used design because higher dilutions would result in only one flow rate combination and, therefore, due to the setup, in only five different distances.

The limitation in the distances is due to the used microscope stage. The flow rate in the detection area is limited, because too high flow rates result in too short bursts, because the molecule is flushed too fast through the focal volume and, therefore, the number of detected photons has a too low statistic.

Figure 7.12: Spontaneous refolding of DM-MBP. DM-MBP was unfolded in 3 M GuHCl.

(A) The GuHCl concentration was diluted to 2 M and then mixed in the mi-crofluidic device 1 to 12 with a buffer containing 20 mM KCl to a final concen-tration of 0.15 M GuHCl. (B-C) The GuHCl concenconcen-tration was diluted to 1.8 M after denaturation. At a mixing ratio of 1 to 12 in the microfluidic device, the concentration of GuHCl was reduced to 0.13 M in the presence of (B) 20 mM KCl or (C) 200 mM KCl. The concentration of DM-MBP was chosen as 15 pM after mixing.

0.15 M GuHCl and 20 mM KCl

unfolded

33 ms

50 ms

155 ms

232 ms

343 ms

512 ms

530 ms

718 ms

792 ms

1.07 s

refolded

unfolded

33 ms

50 ms

155 ms

232 ms

343 ms

512 ms

530 ms

718 ms

792 ms

1.07 s

refolded

0.13 M GuHCl and 20 mM KCl

unfolded

33 ms

50 ms

155 ms

232 ms

343 ms

512 ms

530 ms

718 ms

792 ms

1.07 s

refolded

0.13 M GuHCl and 200 mM KCl

A B C

Figure 7.12A depicts the time dependent changes in the FRET efficiency after dilution from 2 M to 0.15 M GuHCl. A reliable PDA analysis cannot be performed due to the high flow rates and, therefore, very narrow bursts with a too low number of photons. The FRET effi-ciency changes from the unfolded DM-MBP (FRET effieffi-ciency ~0.1) to an intermediate FRET efficiency of ~0.65 after approximately 800 ms. Within the maximum timescale, we can reach with the used microfluidic design, the DM-MBP does not change from the intermediate con-formation to its finally folded concon-formation. Thus, the timescale for this last refolding step is much longer than 1 s.

It is known from literature that the refolding of DM-MBP depends on the GuHCl concen-tration [Chakraborty et al., 2010]. To test if small changes in the final concenconcen-tration have an effect on the refolding kinetics, the maximum concentration, which is needed to keep the DM-MBP unfolded, was verified. We tested the conformation by the FRET efficiency of equilibrium measurements at a GuHCl concentration of 1.8 M and 1.5 M and compared these measurements with the one with a final GuHCl concentration of 2 M (Figure 7.11, dark yellow and red line). The FRET efficiency of the measurements with 1.8 M GuHCl is similar to the FRET efficiency with 2 M. At a concentration of 1.5 M GuHCl, the FRET efficiency shifts to slightly lower values, which is due to the fact that the degree of refolding and unfolding depends first on the GuHCl concentration and than on the time. Therefore, 1.8 M GuHCl was used, because it is the lowest possible GuHCl concentration at which the DM-MBP stays totally unfolded.

For the following measurements, we first denatured the DM-MBP in 3 M GuHCl and then diluted the concentration to 1.8 M GuHCl in 5 mM Tris (pH 7.8), 20 mM KCl, 5 mM Mg[OAc]₂ and 0.001 % Tween20. This sample was then mixed in the microfluidic device in a ratio of 1 to 12 with buffer, which results in a final GuHCl concentration of 0.13 M. The time-dependent refolding is given in Figure 7.12B. The refolding kinetics is faster than observed for the measurements with a final GuHCl concentration of 0.15 M. The intermediate state at

~0.65 FRET efficiency was reached after approximately 500 ms. The final, properly folded conformation was again not reached on the timescale of the microfluidic measurements.

In the next step, we investigated the effect of the concentration of KCl on the refolding ki-netics. [Chakraborty et al., 2010] observed that the refolding gets slower by a factor of two when the concentration of KCl is increased from 20 mM to 200 mM. The same measurement as discussed above (finally 0.13 M GuHCl) in the presence of 20 mM KCl was performed in the presence of 200 mM KCl (Figure 7.12C). The timescale for refolding, measured with microfluidics, does not change depending on the tested salt concentration. The intermediate state was again reached in approximately 500 ms after mixing.

To further analyze the data, the FRET efficiency curves are plotted by a Gaussian function.

The mean FRET efficiency value of the fit is plotted versus the time after mixing (Figure 7.13).

It is clearly visible that the timescale of the refolding is different for the two tested GuHCl concentrations. Fitting the curves using an exponential function results in 681 ±609 ms, 509±244 ms and 428 ± 323 ms for 0.15 M GuHCl and 20 mM KCl, 0.13 M GuHCl and 20 mM KCl and 0.13 M GuHCl and 200 mM KCl, respectively. The standard errors of the fit are very large and, thus, are not determinable. However, it shows that the refolding time in the presence of 0.15 M GuHCl and 20 mM KCl is the larger one.

In summary, we measured the refolding of DM-MBP from an unfolded to an intermediate con-formation. Refolding time for this process depends on the GuHCl concentration, but not on the concentration of the KCl. This is contradictory to the literature. There it is found that the refolding is salt dependent and increases with higher salt concentrations [Chakraborty et al., 2010]. We suggested that when the refolding time from the unfolded to the

intermedi-0.15 M GuHCl and 20 mM KCl 0.13 M GuHCl and 20 mM KCl 0.13 M GuHCl and 200 mM KCl

A M [ms]

Figure 7.13: Kinetic of the spontaneous refolding of DM-MBP. The FRET efficiency curves of Figure 7.12 were fitted using a Gaussian function. The mean FRET efficiency of the fit and standard error are plotted versus the time after mixing for DM-MBP refolded in 0.15 M GuHCl and 20 mM KCl (black dots), in 0.13 M GuHCl and 20 mM KCl (red dot) and 0.13 M GuHCl and 200 mM KCl (cyan dots). Fitting the mean FRET efficiency using an exponential curve results in refolding times of 681± 609 ms, 509±244 ms and 428±323 ms, respectively.

ate state is independent of the salt concentration, then refolding from this point onwards to the final conformation has to be salt dependent.