Results Table 4.7: Chemical shift values and line broadening of the phosphate resonances of

Ras(T35A)·Mg²⁺·GDP in the absence and presence of different cyclen derivatives.

α α

αα-phosphate ββββ-phosphate p:l δδ [ppm] δδ ∆ν∆ν∆ν∆ν1/2 [Hz]^a δδδδ [ppm] ∆ν∆ν∆ν∆ν1/2 [Hz]^a

1:0 -10.55 61 -2.03 33

Zn²⁺-cyclen

1:16 -10.32 70 -2.00 35

1:0 -10.55 64 -2.04 33

Cu²⁺-cyclen

1:16 -10.44 69 -2.02 37

1:0 -10.55 68 -2.06 35

cyclen

1:16 -10.51 66 -2.02 35

a the line width was corrected with respect to the exponential filter used in data processing

The effect of metal(II)-BPAs on the inactive conformation of wild type and mutant Ras complexed to Mg²⁺·GDP was studied for the same reasons as in the case of the metal(II)-cyclens. The experiments have been carried out with wild type and mutant Ras·Mg²⁺·GDP. The obtained spectra are given in Figure 4.23.

Figure 4.23: Influence on metal(II)-BPA on Ras complexed to Mg²⁺·GDP. ³¹P NMR spectra of Ras·Mg²⁺·GDP in 40 mM Tris/HCl pH 7.4, 10 mM MgCl2 and 2 mM DTE, 0.2 mM DSS and 5% D2O. (A), (B), (C) 0.48 mM Ras(wt) in the absence and presence of Zn²⁺-BPA and Cu²⁺-BPA, respectively with a molar excess of 16. (E) and (D) 0.57 mM Ras(T35A) in the absence and presence of a 16-fold excess of Cu²⁺-BPA. (F) and (G) 0.71 mM Ras(G12V) in the absence and presence of an 18-fold excess of Cu²⁺-BPA.

All spectra have been recorded at 278K. Experimental data have been filtered exponentially leading to an additional line broadening of 15 Hz.

Chapter 4 Results Ras(wt)·Mg²⁺·GDP was titrated with Zn²⁺- and Cu²⁺-BPA. The resonance of the β-phosphate group is not influenced significantly by the presence of the two compounds concerning both the line width and the chemical shift. For the α-phosphate peak a downfield shift can be observed upon addition of both compounds, with the effect being stronger in the presence of Cu²⁺-BPA. The line width of the α-phosphate signal is also not influenced significantly.

Additionally the Ras mutants T35A and G12V complexed to Mg²⁺·GDP have been titrated with Cu²⁺-BPA. Again in both cases the β-phosphate resonance is not influenced significantly, whereas the signals of the α-phosphate shift downfield. The effects are comparable for both Ras mutants with a chemical shift difference of 0.43 and 0.39 ppm for T35A and G12V, respectively between Ras·Mg²⁺·GDP in the absence and presence of a 16-fold and 18-fold ligand excess, respectively. This is also in good agreement with wild type Ras, were a downfield shift of 0.45 ppm is observed upon addition of a 16-fold excess Cu²⁺-BPA. Significant line broadening can not be observed for both signals. Table 4.8 summarizes the effects of metal(II)-BPA on Ras·Mg²⁺·GDP.

Table 4.8: Chemical shift values and line broadening of the phosphate resonances of Ras(T35A)·Mg²⁺·GDP in the absence and presence of different BPA derivatives.

α α

αα-phosphate ββ-phosphate ββ Ras·Mg²⁺·GDP p:l δδδδ [ppm] ∆ν∆ν∆ν∆ν1/2 [Hz]^a δδ [ppm] δδ ∆ν∆ν∆ν∆ν1/2 [Hz]^a

Cu²⁺ -BPA

1:0 -10.59 47 -1.97 30

wild type

1:16 -10.14 72 -1.95 43

1:0 -10.59 59 -2.04 30

T35A

1:16 -10.16 78 -1.98 44

1:0 -10.42 45 -1.83 29

G12V

1:18 -10.03 70 -1.80 45

Zn²⁺ -BPA

1:0 -10.59 47 -1.97 30

wild type

1:16 -10.46 66 -2.01 33

a the line width was corrected with respect to the exponential filter used during data processing

Chapter 4 Results

4.3 Perturbation of the Ras-Raf-RBD Interaction by Metal(II)-Chelates

4.3.1 General Considerations

As described in section 1.1.5.2 conformational state (1) of active Ras shows drastically reduced affinity for effector molecules. The above characterized metal(II)-chelates have been shown to selectively recognize and stabilize conformational state (1) of active Ras.

Consequently it should also be possible to perturb the Ras-effector interaction by both mutants via stabilization of conformational state (1). This was already shown by ITC, where a drop of the apparent affinity constant of Raf-RBD for Ras(wt)·Mg²⁺·GppNHp in the presence of Zn²⁺-cyclen and Cu²⁺-cyclen is observed (Rosnizeck et al., accepted). No data are available about the association kinetics between Ras and Raf-RBD in the presence of both compounds. Additionally their mode of inhibition is not known. Since Raf-RBD can bind both conformational states of active Ras the question remains, whether a heterotrimeric complex is formed consisting of Ras·Mg²⁺·GppNHp in conformational state (1), Raf-RBD and Zn²⁺-cyclen or whether the effector and the metal(II)-chelate bind separately to Ras. To address these questions the Ras-Raf-RBD association in the presence of the two compounds was studied further by ³¹P NMR spectroscopy.

4.3.2

P NMR Titration of Ras(T35S)·Mg

·GppNHp Complexed to Raf-RBD with Zn

-Cyclen and Zn

-BPA

The partial loss-of-function mutant Ras(T35S)·Mg²⁺·GppNHp predominately exists in the weak effector-binding state. Upon addition of effector the signals corresponding to conformational state (2) appear in the corresponding ³¹P NMR spectrum. The signals representing conformational state (1) and (2) for the γ-phosphate group in Ras(T35S)·Mg²⁺·GppNHp are very well separated allowing the determination of the proportion of the single states quite easily via the peak areas. Ras(T35S)·Mg²⁺·GppNHp was complexed to Raf-RBD and the displacement of the latter was studied by ³¹P NMR titration with the two Zn²⁺-chelates. Figure 4.24 shows the results obtained with Zn²⁺-cyclen. The three phosphate groups in Ras(T35S)·Mg²⁺·GppNHp are represented by one signal each. Upon addition of Raf-RBD the equilibrium is almost completely shifted towards the effector-binding state. The line widths of the signals representing conformational state (2) are broadened by a factor of 1.43, 1.41 and 1.44 for the α-, the β- and the γ-phosphate peak in complex with Raf-RBD compared to conformational state (1). This relates very well with the increase of the molecular mass by a factor of 1.46, when effector is bound. To the present complex between Ras(T35S)·Mg²⁺·GppNHp and Raf-RBD Zn²⁺-cyclen was added up to a 48-fold molar excess. As one can see very clearly

Chapter 4 Results