Comparison of Different Buffers and their Effects on Intermediate Filaments
4.2 Influence of the Buffers on Tetrameric Protein
The tetrameric state (meaning no assembly reagents added) as well as the assembled filament state of vimentin (addition of 100 mM KCl) are investigated using AFM and SAXS. With AFM the overall filament structure can be observed, whereas SAXS yields information of the protein radius. While in PB the formation of filaments is initiated by the addition of 100 mM KCl, the standard protocol for TRIS proposes a change of the pH from 8.4 to 7.5, an increase of the buffer concentration from 5 mM to 25 mM and the addition of 50 mM NaCl [8]. However, to make TRIS experiments more comparable to PB and MOPS, TRIS is kept at 2 mM or 20 mM concentration and the pH is not changed during the experiments shown here. Assembly is initiated by adding 100 mM KCl, likewise to PB and MOPS experiments.
4.2 Influence of the Buffers on Tetrameric Protein
At first, the tetrameric state of vimentin is investigated in all three buffers at the low and high buffer concentration. The freshly dialyzed protein is prepared for AFM experiments and the
re-(a)
low
high
PB
TRIS MOPS
(b) (c)
(d) (e) (f)
5 µm
Figure 4.1:Typical AFM images of tetrameric vimentin in different buffers: vimentin in 2 mM (a) TRIS, (b) PB and (c) MOPS buffer, each at pH 7.5. Vimentin at the high buffer concentration of 20 mM is shown in (d) TRIS, (e) PB, (f ) MOPS buffer. Whereas nothing is visible at the low buffer concentration, small (roughly 200 nm long) vimentin aggregates are visible in the high buffer concentration.
64 Chapter 4. Comparison of Different Buffers and their Effects on Intermediate Filaments sults are presented in Fig. 4.1.
In Fig. 4.1 (a-c) vimentin protein in the three different buffers (TRIS, PB and MOPS) at low buffer concentration is shown. No aggregates are visible in any of the images, indicating that the struc-tures in those solutions are smaller than what can be detected. In contrast, at high buffer con-centration small structures, most likely aggregated or assembled vimentin protein, can be ob-served in all buffer systems. In the images, it seems that in TRIS and PB (Fig. 4.1d and e) more aggregates are visible than in MOPS buffer (Fig. 4.1f ).
In addition to AFM experiments, SAXS measurements are performed three to six times per buffer and an example of all measurements at one condition are shown in Fig. 4.2.
Exp. 2 Exp. 1 Exp. 3 Exp. 5 Exp. 4 Exp. 6 100
10-2
10-4
I (a.u.)
q (nm-1)
0.1 0.5 1 2
Figure 4.2:Scattering profiles of vimentin filaments assembled with 100 mM KCl in 2 mM MOPS buffer. In total six different measurements are performed and the scattering profiles retrieved.
In Fig. 4.2 the scattering profiles of vimentin filaments assembled with 100 mM KCl in 2 mM MOPS buffer are shown. For this experiment, six different experiments are conducted. All data are analyzed separately, however only one typical SAXS profile of vimentin protein in each buffer is shown in Fig. 4.3. An average is not calculated as the experiments are performed on different days and slight changes in the sample to detector distance are observed, which lead to a differentq-range.
When looking at the scattering profiles of vimentin protein in the three buffers, it can be ob-served that there is no difference between the signals in the three buffers at low concentration, however the profiles of vimentin protein at high buffer concentrations are very different com-pared to the scattering profiles at low buffer concentration. The scattering curves of vimentin protein at low q-values are higher in intensity for the high buffer concentration than for
vi-4.2. Influence of the Buffers on Tetrameric Protein 65
PBTRIS MOPS
}
PBTRIS MOPS
}
100
10-2
10-4
I (a.u.)
q (nm-1)
0.1 0.5 1 2
low high
Figure 4.3:Scattering profiles of tetrameric vimentin at low (2 mM) and high (20 mM) buffer concentration. Three different buffers (TRIS, PB and MOPS) are tested. Scattering profiles of vimentin in the low buffer concen-tration are very similar, however in the high buffer concenconcen-tration the scattering profiles look different.
mentin in the low buffer concentration. For the high TRIS and PB buffer concentration the in-tensity is even higher than for vimentin protein in the high MOPS concentration. The vimentin curves in the high buffer concentrations are steeper than those in the low buffer concentra-tion, indicating that the protein radius increases. By performing a Guinier analysis of the data at small q, the radius of gyration for elongated objectsRc as well as theI(0) value can be ex-tracted (Equation 2.27). Guinier analysis is performed on all data and the results forRc andI(0) are shown in Fig. 4.4.
For the low buffer concentration, vimentin protein in all buffers has an average value ofRc ≈ 2.4 nm, whereas for the high buffer concentration the radius of the vimentin protein varies from Rc ≈4.1 nm in MOPS toRc≈6.2 nm in TRIS buffer (Fig. 4.4a). TheRc values found for vimentin protein in low buffer concentration are in agreement with the literature [7]. The increase in the radius for vimentin at the higher buffer concentration is already seen in the AFM images, where under these conditions small structures, most likely vimentin filaments or aggregates were ob-served.
Fig. 4.4b shows theI(0) values extracted from the Guinier fits. HavingI(0) on arbitrary unit scale, the values can not be used to compute the molecular weight, however the values can be com-pared with each other. The values for vimentin at the low buffer concentration are all very sim-ilar. On the contrary, the I(0) values for vimentin protein in the high buffer concentration are different, following the same trend as theRc values (TRIS has the highest value and MOPS the lowest).
66 Chapter 4. Comparison of Different Buffers and their Effects on Intermediate Filaments
0.03
0.02
0.01
0.00 TRIS PB MOPS TRIS PB MOPS
I(0) (a.u.)
Figure 4.4:Guinier analysis of the scattering profiles of vimentin in the the three buffers (TRIS, PB and MOPS) at low (2 mM) and high (20 mM) concentration. (a) Radius of gyration (Rc) for vimentin in the three different buffers at low and high buffer concentration. (b) I(0)retrieved from the analysis for vimentin in the six different conditions. Similar Rcand I(0)values are found at the low buffer concentration. Different values are retrieved for the three buffers at the high buffer concentration.