active centre and the peptide was estimated and the peptide was thereupon linked to both metal(II)-chelates at the chair of Prof. König. Figure 4.40 shows the designed bivalent ligands.
Figure 4.40: Designed bivalent ligands for Ras. Both Zn2+-chelates have been linked to the small peptide LGGIR giving the hybride ligands H181 (above) and RHC-5-1 (bottom, n = 3) and RHC-6-1 (bottom, n = 2).
4.5.2 Determination of the Affinity of the Bivalent Ligands by STD NMR
The designed hybride ligands carry a peptide moiety and consequently the on-resonance irradiation frequency in the STD experiment has to be chosen carefully as already described in section 4.1.2. It has been shown that it is possible to saturate Ras entirely and selectively at -2 ppm. This frequency should be far enough from the signals from the peptides. However to make sure that reliable results are obtained the compounds were initially measured without Ras being present. In the next step Ras(T35A)·Mg2+·GppNHp was added to the sample and the same experiment was carried out followed by the titration with H181, RHC-5-1 and RHC-6-1 up to a molar excess of 333, 480 and 190, respectively.
Peaks representative for the metal(II)-chelate moiety of the molecule have been used for evaluation. Figure 4.41 and 4.42 show the obtained plot of the amplification factor against the concentration of the hybride ligand carrying either a Zn2+-cyclen or a Zn2+-BPA moiety. The binding curve does not reach saturation at molar ratios between Ras and the hybride ligands of 333, 480 and 190. From these data is it clear that the designed hybride ligands do not have higher affinity for Ras than the metal(II)-chelates alone.
Chapter 4 Results
0 2 4 6 8 10 12
0 1 2 3 4 5
c (ligand) [mmol L-1] ASTD
Figure 4.41: Plot of the STD amplification factor against the molar excess of Zn2+-cyclen and the hybrid ligands RHC-5-1 and RHC-6-1. Shown are the amplification factors obtained from a STD titration with a sample originally containing 50 µ M Ras(T35A)·Mg2+·GppNHp in 40 mM Tris/HCl pH 7.4 and 10 mM MgCl2 with Zn2+-cyclen (red dots), RHC-5-1 (blue dots) and RHC-6-1 (black dots), respectively. In each experiment on-resonance irradiation was performed at -2 ppm and a spinlock filter was implemented (50 ms at 15 dB). In the titration of RHC-5-1 the on-resonance irradiation was performed with an attenuation of 30 dB resulting in higher ASTD-values compared to the titrations with Zn2+-cyclen and RHC-6-1 (the attenuation was set to 40 dB) due to the more effective saturation of the protein. The obtained values for ASTD have been multiplied by a factor of 0.1 in the case of RHC-5-1 in order to allow for a better comparison with the data obtained for Zn2+-cyclen and RHC-6-1. All spectra were recorded at 278 K at 600 MHz proton frequency. In each case the signals representing the Zn2+-cyclen moiety have been used for evaluation. The data have been fitted according to Equation 3.2. The corresponding fits are represented by the red (Zn2+-cyclen) and the black line (RHC-6-1). For Zn2+-cyclen a KD-value of 9.65 ± 1 mM is derived. For the RHC-6-1 the KD-value can be estimated to be larger than 40 mM. The data for RHC-5-1 do not allow for an estimation or quantification of the KD-value.
0 1 2 3 4 5
0 4 8 12 16
ASTD
c (ligand) [mmol L-1]
Figure 4.42: Plot of the STD amplification factor against the molar excess of Zn2+-BPA and the hybrid ligand H181. Shown are the amplification factors obtained from a STD titration with a sample originally containing 50 µM Ras(T35A)·Mg2+·GppNHp in 40 mM Tris/HCl pH 7.4 and 10 mM MgCl2 with Zn2+-BPA (red dots), RHH 181 (black dots), respectively. The on-resonance irradiation was set to -2 ppm with an attenuation of 30 dB (H181) and 0.3 ppm with an attenuation of 40 dB (Zn2+-BPA). A spinlock filter was implemented (50 ms at 15 dB) in order to suppress protein proton resonances potentially overlapping with the ligand signals. The data obtained in the titration with Zn2+-BPA have been fitted according to Equation 3.2 giving a KD-value of 2.07 ± 0.25 mM. The corresponding fit is represented by the red line. The data for H181 do not allow for an estimation or quantification of the KD-value. The liner regression of the data is shown by the dashed black line.
Chapter 4 Results
4.6 Characterization of the Interaction between Ras and Peptides Derived from Raf-RBD
4.6.1 Localization of the Binding Site in Ras(wt) · Mg
2+· GppNHp and Ras(T35A) · Mg
2+· GppNHp
In section 1.1.6.3 peptides derived from the Ras binding domain of the Ras effector Raf and their modified analogues have been described, which potently interfere with the Ras-Raf association (Barnard at al.1995 1998). The peptide CCAVFRL compromising Raf-RBD residues 95-101 and the peptide CCFFFRRL obtained from computational design were investigated in more detail by NMR spectroscopy since the binding position of these peptides and also their mode of inhibition have not been elucidated so far. For that reason the two peptides have been subject to chemical shift perturbation studies with both wild type Ras and the T35A mutant complexed to Mg2+·GppNHp. For the wild type mutant an assignment was only available for pH 5.5 at this point of time (Ito et al. 1997). In order to allow for a good comparison all investigations have been carried out at this pH value.
CCAVFRL and CCFFFRRL were first titrated to wild type Ras complexed to Mg2+·GppNHp up to a molar excess of 4 and 3.2, respectively and the effect was followed by [1H, 15N]-HSQC spectroscopy. Figure 4.43 shows the combined chemical shift changes observed in wild type Ras·Mg2+·GppNHp in the presence of the two peptides.
Figure 4.43: Combined chemical shift changes observed in wild type Ras·Mg2+·GppNHp in the presence of the peptide CCAVFRL (left panel) and CCFFFRRL (right panel). Given are the chemical shift changes observed in a sample originally containing 1.2 mM 15N c´Ras(wt)·Mg2+·GppNHp in 20 mM Na2HPO4-NaH2PO4 pH 5.5, 10 mM MgCl2, 2 mM DTE, 0.2 mM DSS and 5% D2O upon addition of a 4-fold and 4.8-fold excess of the peptides CCAVFRL and CCFFFRRL dissolved in the same buffer, respectively. All spectra have been recorded at 293 K at 800 MHz proton frequency. The corrected standard deviation to zero (σ0corr
) is given by the blue line (Schumann et al. 2007).
Chapter 4 Results