I NVESTIGATION OF DIVALENT LIGAND 41 WITH BIFREQUENCY CW -EPR SPECTROSCOPY

The EPR experiments in this chapter were performed and evaluated by Dipl. Chem. P. Braun.

An increase in the ligand concentration of 41 (Figure 26) with constant WGA concentration leads in theory to a shift of the equilibrium from chelatingly bound ligand (C1) to monovalently bound divalent ligand (C3) (Figure 26) and the EM could be determined through the ligand concentration at which the population of the chelatingly bound ligand (C1) and the monovalently bound divalent ligand (C3) become equal.

Figure 26: Crystal structure of WGA dimer (PDP code: 2X52) with subunits colored in blue and green. Primary binding sites are marked through white framed brown dots and spin labels are depicted through small yellow dots. The left picture shows the schematic chelating binding of a divalent ligand 41 (C1) and the right picture shows monovalent binding of a divalent ligand 41 (C3). The binding mode of the monovalently bound divalent ligand with the spin-labeled GlcNAc residue is not considered, because it can not be distinguished from the chelating binding mode through cw-EPR spectroscopy. Both binding modes are in dynamic equilibrium with unbound ligand in solution (C2).

For an experimental EM determination for the ligand 41 the percentage of the three components C1-C3 were calculated as a function of the ligand concentration using cw-EPR spectroscopy. The cw-EPR spectrum from an excess of ligand 41 with WGA is a superposition of the signals from the three different components C1-C3.

Therefore the three components were determined in separate experiments and the mixed spectra could be simulated through a linear combination of the fitted single spectra. The cw-EPR spectrum from the chelated component (C1) was accessible through measurements using an equimolar ratio of 41 and WGA dimer. At this ratio 41 binds almost quantitative with negligible percentage of C2 and C3. The obtained cw-EPR spectra (black) in X-band (left) and in Q-band (right) are depicted in Figure 27 top. For the spectra simulation (red curves Figure 27) the set of parameters was simplified by considering only the four primary binding sites of WGA.

Since the primary binding sites possess a tenfold stronger binding affinity, binding to the four secondary binding sites was neglected. As for C1, the cw-EPR spectra for C2 were experimentally accessible through

measuring 41 in solution without WGA. The obtained cw-EPR spectra are depicted in Figure 27 bottom. A higher frequency was better suitable for the analyses of fast dynamics. The simultaneous evaluation of both spectra delivered an almost ideal simulation. For the determination of the cw-EPR spectra for C3, monovalent ligand 44 (Figure 26 bottom) was used as a model, since such a binding mode is not expected for 41 until high ligand concentrations. However, according to the ELLA experiments described in chapter 3.1.2, for a monovalent ligand a considerably lower binding affinity was expected compared to a divalent ligand. Thus even with a protein excess the cw-EPR was expected to be an overlay of the cw-spectra from bound 44 and unbound 44.

Figure 27: X-band (left) and Q-band (right) cw-EPR spectra (T = 295 K). Experimental cw-spectra are depicted in black, simulated spectra are depicted in red. Top: cw-EPR spectra of 41 with a ligand to WGA dimer ratio of 1:1. Bottom: cw-EPR spectra from 41 in absence of WGA. With these experiments the parameters for the components C1 and C2 were accessible.

Figure 28 depicts the mobility measurements in X-band (left, red) and in Q-band (right, red) at a ligand/WGA dimer ratio of 1:8. For comparison the cw-EPR spectra of 44 in absence of WGA are depicted in the same graph (black). Between the red and the black curves a clear intensity difference is visible since the measurements with WGA contain apart from a fraction of unbound ligand also a slowed fraction of monovalently bound ligand. This leads to a spectrum with broader line width and smaller signal intensity. For the determination of C3 the signal of the bound fraction was separated from the unbound fraction prior to line shape analysis. A detailed description of the used linear combination can be found elsewhere.^[47]

Figure 28: Top: Comparison of the normed X-band (left) and Q-band (right) cw-EPR spectra (T = 295 K) of 44 at a ligand/WGA dimer ratio of 1:8 (red) and in absence of WGA (black). Bottom: through the simulation, the parameters for C3 were obtained.

After the parameters for all three binding modes C1-C3 had been determined through spectra simulation, the population of the three binding modes could be calculated for any ligand/WGA dimer ratio. Due to the results from previous EPR experiments (chapter 1.4) the shift of the equilibrium from chelatingly bound ligand (C1) to monovalently bound divalent ligand (C3) (Figure 26) is not expected until a large excess of ligand concentration.

Therefore, samples with a ligand 41/WGA dimer ratio of 4:1, 6:1, 8:1, 12:1 and 16:1 were used for mobility measurements in X-band and in Q-band (experimental spectra can be found in the dissertation from P.

Braun^[47]). The spectra simulation of the experimental spectra delivered the amount of chelatingly bound ligand (C1), unbound ligand (C2) and monovalently bound ligand (C3) for every ligand/WGA dimer ratio which can be found in Table 2.

P. Braun calculated for the fitting results an error of 2 %- 3 %.^[47] The percentage of C3 lies therefore within the error range. Since the binding of the spin-labeled GlcNAc end and the unlabeled GlcNAc end of 41 have an equal probability, which could be shown in the ELLA experiments in chapter 3.1.2, the amount of C3 is twice as high. This factor of two was not considered in the results presented in Table 2. While the percentage of C1 with increasing ligand excess decreases, the percentage of C2 increases continuously up to 72 % at a ligand/WGA dimer ratio of 16:1. In contrast, the percentage of C3 stays nearly unchanged between 1 % and 4 %. This illustrates that the divalent ligand 41 binds also at a ligand/WGA dimer ratio of 16:1 mainly in a chelating

binding mode to WGA without a significant amount of monovalently bound divalent ligand. Samples with a higher ligand excess delivered no reproduceable results, since the strong signal of the unbound ligand prevented the analysis of the bound ligand. The percentage of all binding modes is summarized in Figure 29.

Table 2: Percentage of the three components for ligand 41 obtained at different ligand/WGA dimer ratio.

41/WGA dimer C1 C2 C3

1:0 0 % 99 % 1 %

4:1 75 % 23 % 2 %

6:1 63 % 35 % 2 %

8:1 50 % 46 % 4 %

12:1 35 % 63 % 2 %

16:1 27 % 72% 1 %

Figure 29: Amount of chelatingly bound ligand (C1), unbound ligand (C2) and monovalently bound ligand (C3) in dependancy of the ligand/WGA dimer ratio for ligand 41. In these experiments the concentration of WGA dimer was kept constant and the ligand concentration was varied.

Even though it was not possible to determine EM for ligand 41, the EM could be estimated since we know the ligand concentration with the highest ligand to WGA dimer ratio (16:1, [41] = 800 µM), which was not high enough to reach the shift of the equilibrium from chelatingly bound ligand (C1) to monovalently bound divalent ligand (C3) (Figure 26). Due to the correlation [BB]switch = EM/2 for 41 EM > 1600 µM. This shows also the upper limit of the ligand to protein ratio applicable with EPR experiments.

3.2.2 Investigation of divalent ligand 40 with bifrequency