Results The identical buffer solution was used for the sample, the data were recorded at the same

spectrometer at the same temperature and the same settings for the experiments (M. Spoerner, Regensburg). Subsequently the chemical shifts have been directly compared.

Figure 4.47 shows the chemical shift differences between Ras(T35S) and Ras(T35A) complexed to Mg²⁺·GppNHp as a function of the primary sequence. Not surprisingly the largest differences can be observed for residue 35, which is the mutated one and residues in its close proximity. Residues with an observed combined differences between the chemical shifts ∆δcomb > σ0corr

are given in Table 4.16. Residues affected by the mutation can be found in both switch regions and around helix α3. In Figure 4.48 these residues are mapped on the NMR structure of Ras(T35S)·Mg²⁺·GppNHp (F. Schumann, unpublished results).

Figure 4.47: Chemical shift differences observed between the [¹H, ¹⁵N]-TROSY-HSQC spectra of the Ras mutants T35S and T35A complexed to Mg²⁺·GppNHp. Data have been obtained from experiments under identical conditions. (M. Spörner, University of Regensburg). The samples contained 0.75 mM Ras(T35S)·Mg²⁺·GppNHp and 0.7 mM Ras(T35A)·Mg²⁺·GppNHp in 20 mM NaPi pH 5.5, 10 mM MgCl2, 2 mM DTE, 0.2 mM DSS and 5% D2O. All spectra have been recorded at 293 K. The corrected standard deviation to zero (σ0corr

) is given by the blue line.

Table 4.16: Residues exhibiting a significant difference in the combined chemical shifts between the [¹H, ¹⁵N]-HSQC spectra of the Ras mutants T35A and T35S complexed to Mg²⁺·GppNHp. The mutated residue is given in bold letters.

∆δ∆δ

∆δ∆δ_comb > σσσσ₀^corr

3, 5, 8, 12, 13, 16, 21, 29, 32, 33, 35, 36, 38, 39, 40, 41, 46, 52, 53, 54, 57, 58, 59, 60, 62, 63, 64, 66, 68, 69, 70, 71, 72, 76, 79, 92, 93, 94, 95, 97, 105, 111, 112, 159, 161, 162

Chapter 4 Results

Figure 4.48: Residues showing significant combined chemical changes in the [¹H, ¹⁵N]-HSQC-spectra of Ras(T35A) and Ras(T35S) complexed to Mg²⁺·GppNHp. Residues exhibiting a combined chemical shift difference ∆δcomb larger than the standard deviation to zero are shown in red. Ser³⁵ is shown in green.

Prolines and not assigned residues are shown in gray.

4.8 High Pressure NMR Spectroscopic Investigations on Ras(wt)·Mg

·GppNHp

As described in section 1.1.5.3 under high pressure the dynamic equilibrium in wild type Ras·Mg²⁺·GppNHp is shifted from the strong effector-binding substate (2) towards conformational state (1), which is recognized by GEFs (Kalbitzer et al. 2009). The functional cycle of Ras as described in section 1.1.1 requires the coexistence of more than the two conformational substates (1) and (2), which are sensed by ³¹P NMR spectroscopy by different chemical shifts for the α- and γ-phosphate group (Geyer et al. 1996, Spoerner et al. 2004 2005a 2007). The GAP-binding conformation has not been detected so far and additionally local unfolding states and the completely unfolded state have not been investigated. In order to obtain information about the different conformational substates coexisting in the Ras protein in solution at atomic resolution, the pressure effect was followed using [¹H, ¹⁵N]-HSQC spectroscopy. For a better comparison the 2D experiments have first been carried out at the same experimental conditions with regard to the

31P pressure series, i.e. at a pH value of 7.4 and a temperature of 278 K. Additionally a pressure series at 303 K was recorded. Due to the poor signal to noise ratio achieved in the pressure series at 278 K mainly the experimental data from the pressure series recorded at 303 K have been used for evaluation. [¹H, ¹⁵N]-HSQC spectra have been recorded at 0.3 MPa and up to 200 MPa and 180 MPa, respectively. The pressure series recorded at 303 K was only performed up to a pressure of 180 MPa due to the “explosion” of the ceramic cell at a pressure of 200 MPa. The results are given in the following paragraphs.

Chapter 4 Results

4.8.1 Pressure Dependence of the Chemical Shifts

Increasing pressure results in chemical shift changes of all resonances at both temperatures. The observed shift directions are qualitatively the same at both temperatures.

Figure 4.49 shows the overlay of the [¹H, ¹⁵N]-HSQC spectra of Ras(wt)·Mg²⁺·GppNHp under different pressures at both temperatures.

Figure 4.49: Pressure dependent shifts in Ras(wt)·Mg²⁺·GppNHp: (A) Overlay of the [¹H, ¹⁵N]-HSQC spectra of 1.05 mM Ras(wt)·Mg²⁺·GppNHp in 40 mM Tris/HCl pH 7.4, 10 mM MgCl2, 2 mM DTE, 0.1 mM DSS and 12% D2O recorded at 278 K under different pressures between 3 and 200 MPa.

(B) Overlay of the [¹H, ¹⁵N]-HSQC spectra of 1.84 mM Ras(wt)·Mg²⁺·GppNHp in 40 mM Tris/HCl pH 7.4, 10 mM MgCl2, 2 mM DTE and 12% D2O recorded at 303 K under increasing pressures of 3 MPa and 180 MPa. The direction of chemical shift with increasing pressure is indicated by the arrows. All spectra have been recorded at 800 MHz proton frequency.

In general the chemical shift changes induced by the local and global conformational changes are a non-linear function of the pressure. The chemical shift changes induced by pressure can be described by a 2^nd order Taylor expansion according to Equation 3.4 (see section 3.3.4.3). The pressure effects are composed of two contributions, an unspecific effect, which is also observed in random coil peptides and a specific effect, characteristic for a certain protein. Consequently the chemical shifts of the amide protons were corrected by the known pressure response of random-coil peptides (Arnold et al. 2002) and fitted according to Equation 3.4. Unfortunately the corresponding information for the correction of the nitrogen shifts in random-coil peptides is missing, but work is currently going on at the department. The first order conformation dependent pressure coefficients |B1* (H^N)|

and |B1 (N)| determined for the single residues in wild type Ras complexed to Mg²⁺·GppNHp at 303 K are depicted in Figure 4.50 as a function of the sequence position.

Figure 4.51 shows the second order coefficients |B2*(H^N)| and |B2 (N)|, respectively.

Chapter 4 Results

Figure 4.50: First order pressure coefficients of wild type Ras·Mg²⁺·GppNHp derived from a pressure series at 303 K. Shown are the absolute values of the first order pressure coefficients of H^N (corrected by the standard values derived from model peptides) (A) and N (B) as a function of the primary sequence. The mean value (straight line) and the mean value plus /minus the standard deviation (dashed lines) are shown. Prolines are marked with P, not assigned or not assignable residues, respectively with * and residues, for which the coefficients could not be determined due the loss of signal intensity within the pressure series are marked with I.

Chapter 4 Results

Figure 4.51: Second order pressure coefficients of wild type Ras·Mg²⁺·GppNHp derived from a pressure series at 303 K. Shown are the absolute values of the second order pressure coefficients of H^N (corrected by the standard values derived from model peptides) (A) and N (B) as a function of the primary sequence. The mean value (straight line) and the mean value plus / minus the standard deviation (dashed lines) are shown. Prolines are marked with P, not assigned or not assignable residues, respectively with * and residues, for which the coefficients could not be determined due the loss of signal intensity within the pressure series are marked with I.

Chapter 4 Results