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. 2023 Aug 15:4:e6.
doi: 10.1017/qrd.2023.4. eCollection 2023.

On the micelle formation of DNAJB6b

Andreas Carlsson  1 , Ulf Olsson  2 , Sara Linse  1
Affiliations

On the micelle formation of DNAJB6b

Andreas Carlsson et al. QRB Discov. .

Abstract

The human chaperone DNAJB6b increases the solubility of proteins involved in protein aggregation diseases and suppresses the nucleation of amyloid structures. Due to such favourable properties, DNAJB6b has gained increasing attention over the past decade. The understanding of how DNAJB6b operates on a molecular level may aid the design of inhibitors against amyloid formation. In this work, fundamental aspects of DNAJB6b self-assembly have been examined, providing a basis for future experimental designs and conclusions. The results imply the formation of large chaperone clusters in a concentration-dependent manner. Microfluidic diffusional sizing (MDS) was used to evaluate how DNAJB6b average hydrodynamic radius varies with concentration. We found that, in 20 mM sodium phosphate buffer, 0.2 mM EDTA, at pH 8.0 and room temperature, DNAJB6b displays a micellar behaviour, with a critical micelle concentration (CMC) of around 120 nM. The average hydrodynamic radius appears to be concentration independent between ∼10 μM and 100 μM, with a mean radius of about 12 nm. The CMC found by MDS is supported by native agarose gel electrophoresis and the size distribution appears bimodal in the DNAJB6b concentration range ∼100 nM to 4 μM.

Keywords: affinity; aggregation; chaperone action; oligomers; self-association.

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Figures

None
Graphical abstract
Figure 1.
Figure 1.
Right: model of the DNAJB6b structure as predicted using Alpha Fold, with the N-terminal domain coloured red, the C-terminal domain blue and the linker region grey. Left: chemical structures of the two fluorophores IRdye680 (Li-Cor, 2023), and Alexa647 (Gebhardt et al., 2021) shown at the same scale as the DNAJB6b model.
Figure 2.
Figure 2.
Schematic illustration of the MDS technique. Fluorescence detection is used to measure the fraction of protein that has ended up in chamber A versus B. Since only diffusion causes particles to move in the cross-flow direction, the intensity ratio between chamber A and B can be related to the average hydrodynamic radius of the particles.
Figure 3.
Figure 3.
(a) ⟨RH⟩ of DNAJB6b at various times after dilution, obtained using MDS. The blue series represents Alexa647-labelled protein diluted from 1.4 μM to 20 nM, measured in quadruplicates for each flowrate (setting 2 and 3) and time. The standard deviations are shown as errorbars. The non-diluted sample is shown as a blue square at time zero. Non-labelled DNAJB6b (black), diluted from 6 μM to 100 nM, was measured in a minimum of triplicates for each time point. The grey square represents the ⟨RH⟩ of the non-diluted sample. Exponential decay functions (dashed lines) on the form f(x) = a ∗ e−t/b  + c are fitted to each series. The labelled protein is described by a = 3.2 nm, b = 2.3 days, and c = 4.7 nm. Non-labelled protein is described by a = 5.2 nm, b = 1.0 days, and c = 4.3 nm. (b) Total fluorescence intensities of 20 nM Alexa647-DNAJB6b during 31 h after dilution, measured with MDS. The sample volumes used during storage in each container were 5 mL (blue) and 100 μL (red), providing two highly different surface-area-to-volume ratios.
Figure 4.
Figure 4.
⟨RH⟩ of DNAJB6b measured with MDS as a function of protein concentration, plotted with linear (a) and logarithmic (b) x-axis. Blue data points show measurements for samples containing 20 nM Alexa647-DNAJB6b and varying amount of non-labelled protein to total concentrations ranging between 20 nM and 100 μM. Each data point represents a mean ⟨RH⟩ of several flowrates and times since dilution. For more details regarding data collection, see Methods and Supplementary material. In black, non-labelled DNAJB6b at 100 nM–28 μM. All samples were measured in at least triplicates (except for 28 μM non-labelled protein, which is a singlicate). The errorbars represent the standard deviations of the replicates. The red lines describe the concentration independent region below 100 nM and a linear fit of the radius versus the logarithm of the concentration in the range 160 nM–2 μM. The two lines intersect at 120 nM.
Figure 5.
Figure 5.
Native agarose gel electrophoresis of 5 nM IR-labelled DNAJB6b together with various concentrations of non-labelled DNAJB6b, with total concentrations as indicated by different colours, see full legend in panel b. Panel a shows a scan of the blotted gel, obtained using an IR-fluorescence scanner. Analysis of the IR intensities along the direction of movement gives an electrophoretic mobility profile for each sample, in arbitrary length units, l. u. All profiles are plotted in panel b with shifted baselines. The profiles are superimposed with the gel, in pale colors, rotated and expanded along the direction of the applied voltage relative to panel a, so that pixels and electrophoretic mobility are directly translatable. In panel c, the profiles of 37 nM–4 μM DNAJB6b are plotted with a common y-axis, to compare the profiles where the shift in electrophoretic mobility occurs.
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