4.2.1 Qualitative DNA binding analysis

To asses possible DNA binding, a qualitative EMSA was performed with the purified proteins. The same binding buffer conditions were used, as previously desribed for GCN4 and rGCN4 (Hollenbeck et al. 2001). DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein (Figure 4).

Figure 4: Qualitative EMSA DNA binding 0.5 µM of annealed oligos (20 bp) containing either one CRE or INVii binding site were incubated with 200 µM of protein and equilibrated in binding buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂HPO₄, 1.8 mM KH₂HPO₄, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA). Gel electorphoresis was performed with a pre-equilibrated TGX-Gel in TBE running buffer. Gel-bands were visualized by staining with SYBR Safe and imaged with an UV transilluminator.

4.2.2 Quantitative DNA binding analysis

To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4. Here 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:

Θ_app = Θ_min + (Θ_max - Θ_min) × (K_a² [L]_tot²) / (1 + K_a² [L]_tot²) Equation 1

Here [L]_tot describes the total protein monomer concentration, K_a corresponds to the apparent monomeric equilibration constant. The Θ_min/max values are the experimentally determined site saturation values (For this experiment 0 and 1 were chosen for min and max respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation constant K_k of (0.2930.033)×10^-6 M, which is almost identical to the rGCN4 binding affinity to INVii a _d of (0.2980.030)×10^-6 M.

Figure 5: K_d Calculation of GCN4 and rGCN4 Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of varying concentrations were incubated with 0.5 µM DNA (20 bp, containing one binding site for CRE or INVii) in Binding buffer 1, and the bound fraction analyzed by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction using ImageJ. At least three separate measurements were conducted for each data point. Values are presented as mean +/- SD

To better understand the proteins, quantitative analysis was done to determine the apparent dissociation constant for GCN4 and rGCN4.
For the purified proteins GCN4, rGCN4 and the fusion bGCN4 a qualitative gel was run with high protein concentration (Figure 3). Bands for GCN4 and rGCN4 were visible but no band for bGCN4 could be detected, indicating a lack of DNA binding. This suggests that the dimerization, necessary for DNA binding, is disrupted by the GSG-linker (Ellenberger et al., 1992; Liu et al., 2006; Lupas et al., 2017; Woolfson, 2023). To better understand possible problems in dimerization circular dichroism can be used to analyze secondary structure and proper coiled coil formation (Greenfield, 2006). Further engineering can be done by testing out various linkers with specific properties to ensure correct folding and dimerization (Chen et al., 2013). The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10^-6 M) and rGCN4 ((0.2980.030) × 10^-6 M) are approximately a factor 10 higher then those described in literature ((96) × 10^-8 M for GCN4 and (2.90.8) × 10^-8 M for rGCN4) (Hollenbeck et al., 2001). The differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.

The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed