time scale prevail only a single cross peak is observed for the free and complexed form of a given residue with a chemical shift at the population averaged position. All these effects have to be monitored very accurately in order to prevent errors during data evaluation (see next paragraph). If fast exchange is present the combined chemical shift changes (Schumann et al. 2007) can directly be monitored as a function of the ligand concentration.
In the case of slow and intermediate exchange signal intensities or the line widths, respectively have to be evaluated. In the first case the relative signal intensity of the new cross peak representing the bound conformation is normalized for the overall signal intensity of both cross peaks belonging to the same residue. In the case of intermediate exchange the change of the line width depending on the ligand concentration is consulted for evaluation. Each NMR parameter can be used in order to determine the binding affinity by plotting the change against the ligand concentration.
1.2.4 Paramagnetic Relaxation Enhancement
Paramagnetic systems, such as transition metal complexes or free radicals contain at least one unpaired electron. Both the chemical shift and the line width of NMR active nuclei can be affected through the interaction with the unpaired electron spins. Consequently paramagnetic substances are subdivided into shift and relaxation reagents, respectively.
The latter ones accelerate the relaxation rates of the NMR active nuclei, a phenomenon known as paramagnetic relaxation enhancement (PRE), originally described by Solomon (1955). As a consequence the corresponding signals in the NMR spectrum are strongly broadened depending on the distance to the paramagnetic centre. This effect can be so strong that the signals disappear in the noise level and they are so-called ‘bleached”. The transition metal ion Cu2+ exhibits a very fast relaxation rate. Proton signals in a distance less than 11 Å are affected (Arnesano et al. 2003). For that reason the binding sites of the small organic molecule metal(II)-cyclen, which is known to selectively stabilize the weak binding state in active Ras (see section 1.1.6.4) has been investigated in titration studies
using the paramagnetic derivative Cu2+-cyclen (T. Graf 2006).
As described in paragraph 1.2.3.1 line broadening is not only caused by the presence of a paramagnetic centre, but can also be observed, when exchange processes, which are slow or intermediate compared to the NMR time scale occur. For that reason data from binding studies using a paramagnetic derivative of the ligand have to be evaluated taking these possible effects into account in order to give reliable results. The best approach herein is to directly compare the data with the ones obtained in titration studies with a diamagnetic derivative of the ligand. Line broadening observable in the presence of both derivatives cannot clearly be assigned to the distance-dependent paramagnetic effect, but may also represent structural changes induced by the binding of the ligand elsewhere in the protein.
Chapter 2 Materials
2 MATERIALS
2.1 Chemicals
The basic equipment in the laboratory is purchased from the companies Fluka (Neu-Ulm, Germany), Merck (Darmstadt), Roche (Mannheim), Novabiochem (Läufelingen, Switzerland), Acros Organics (Geel, Belgium), Roth (Karlsruhe), Pharma-Waldorf (Düsseldorf) and Sigma (Deisenhofen) with analytical grade.
The metal(II)-cyclen and metal(II)-derivatives and the peptide LIGGR have been synthesized at the chair of Organic Chemistry (Prof. B. König) at the University of Regensburg.
The peptides with consensus Ras binding sequences (CCAVFRL, CCFFRRRL) have been synthesized by the company GenScript Corporation (Piscataway, USA) with purity between 95% and 98%.
2.2 Enzymes
Alkaline Phosphatase Boehringer/Roche (Mannheim, Germany)
DNase I Boehringer/Roche (Mannheim, Germany)
Lysozyme Sigma (Deisenhofen, Germany)
Thrombin Sigma (Deisenhofen, Germany)
2.3 Frequently Used Buffer Solutions
Buffer A 32 mM Tris/HCl pH 7.4, 10 mM MgCl2, 1 mM DTE Buffer B 64 mM Tris/HCl pH 7.4, 10 mM MgCl2, 400 mM NaCl,
2 mM DTE, 0.1 mM GDP
Buffer C 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM DTE, 1 mM PMSF
Buffer D 50 mM Tris/HCl pH 7.4, 150 mM NaCl, 5 mM DTE Buffer E 40 mM HEPES/NaOH pH 7.4, 10 mM MgCl2,
150 mM NaCl, 2 mM DTE
Buffer F 40 mM Tris/HCl pH 7.4, 10 mM MgCl2, 2 mM DTE
Chapter 2 Materials
2.4 Plasmids
For the expression of the different truncated (aa 1 - 166) and full length (aa 1 - 189) human H-Ras proteins ptac-vectors have been used. The expression of the Ras binding domain of Raf-kinase (aa 51-131) was carried out using pGEX-4T-vectors.
2.5 Bacteria Strains
Escherichia coli CK600K supE, hsdM+, hsdR-, kanR (Hoffmann-Berling,
Heidelberg, Germany)
Escherichia coli BL21(DE3) F-, opmT, hsds (rB
-, mB
-), gal (38,39) (Stratagene, Heidelberg, Germany
2.6 Media and Antibiotics
Luria-Bertani (LB)-medium 10 g bacto-tryptone 5 g yeast extract
10 NaCl
were filled up to 1 L with millipore H2O. After adjusting the pH to 7.0 by 5 mmol L-1 NaOH the medium was autoclaved.
SL-6 stock solution 100 mg ZnSO4 x 7 H2O 30 mg MnCl2
300 mg HBO3
200 mg CoCl2 x 6 H2O 10 mg CuCl2 x 2 H2O 20 mg NiCl x 6 H2O 30 mg Na2MoO4
were filled up to 1 L with millipore water and autoclaved. The stock solution can be stored for long-term at 4 °C.
SL-4 stock solution 500 mg EDTA
200 mg FeSO4 x 7 H2O
were filled up to 90 mL with millipore water. This solution has to be prepared freshly.
Chapter 2 Materials
SL-mix 1 mL SL-6 stock solution
0.9 mL SL-4 stock solution
were filled up to 10 mL with millipore water and sterile filtered.
Minimal medium 7.5 g NaHPO4
3 g KH2PO4
0.5 g NaCl
0.25 g MgSO4 x 7 H2O 0.014 g CaCl2 x 2 H2O
were filled up to 900 mL with millipore water and autoclaved.
New minimal medium (NMM) 900 mL minimal medium 10 mL SL-mix
4 g glucose
1 g NH4Cl
were filled up to 1 L with sterile millipore water.
Each antibiotic was purchased from GERBU Biotechnik GmbH (Gaiberg, Germany).
2.7 Protein Standard
SDS7
(66/45/36/29/24/20.1/14.2 kDa) Sigma (Deisenhofen, Germany)
2.8 Expendable Materials
Bradford reagent Biorad (München, Germany) NMR sample tubes
5 mm Norell Inc. (Landsville, NJ, USA)
8 mm Shigemi Co. LTD (Tokyo, Japan)
Quart glass cells Perkin Elmer (Waltham, MA, USA) Vivaspin ultrafiltration units Vivascience (Hannover, Germany)
Chapter 2 Materials
2.9 Columns
Nucleosil 100 C18 precolumn Bischoff Cromatography (Leonberg, Germany) ODS hypersil C18 column Beckman Coulter (Fullerton, CA, USA) Q-sepharose Amersham Pharmacia (Freiburg, Germany) Q-sepharose fast flow Amersham Pharmacia (Freiburg, Germany) Superdex G-75 prep grade Amersham Pharmacia (Freiburg, Germany)
2.10 Instruments
Beckman HPLC-system Gold Beckman (München, Germany)
FPLC-System Amersham Pharmacia (Freiburg, Germany)
Luminescence spectrometer LC-50 Perkin Elmer (Waltham, MA, USA) NMR spectrometer
Avance 500 Bruker (Karlsruhe, Germany)
Avance 600 Bruker (Karlsruhe, Germany)
Avance 800 Bruker (Karlsruhe, Germany)
Ultrasonic device Sonifier 250/450 Branson (Schwäbisch Gmünd, Germany) UV/Vis-Spectrometer Uvikon 933 Kontron (Neufahrn, Germany)
2.11 Data Analysis and Graphical Software
Auremol 2.4.0 www.auremol.de
CorelDraw 12 Corel Corporation (Ottawa, Canada)
Origin 6.0 Microcal Software Inc. (Northampton, MA, USA)
PyMOL DeLano Scientific (San Francisco, CA, USA)
Topspin 2.0 Bruker (Karlsruhe, Germany) ViewerLite 4.2 Accelrys Inc. (San Diego, CA, USA)
Chapter 3 Methods
3 METHODS
3.1 Molecular Biology
3.1.1 Expression and Purification of Unlabeled Ras Proteins
Overexpression of human H-Ras was performed according to Tucker et al. (1986) by using ptac-vectors in the E. coli strain CK600K. 10 L of the standard LB medium containing 100 mg L-1 carbenicilline and 25 mg L-1 kanamycin was inoculated with 100 mL of an overnight culture. Incubation was carried out at 37 °C and a shaker speed of 180 rpm.
Expression was induced at an OD600-value between 0.8 and 1 with 1 mM IPTG.
Centrifugation of the culture was performed after overnight incubation (about 14 to 18 hours). The precipitate was frozen and stored at -20 °C.
The unfreezed cell pellet was resuspended in cell disruption buffer (32 mM Tris/HCl pH 7.4, 5 mM EDTA, 2 mM DTE and 1 mM PMSF) and cell disruption was initiated by addition of lysozyme (1 mg per mL cell suspension). After 30 minutes of incubation 10 mM MgCl2 and 20 mg DNase I per 100 mL cell suspension were added and the mixture was allowed to incubate another 30 minutes at 4 °C. Sedimentation of the cell fragments was achieved by 60 minutes of centrifugation with 18.000 g. The clear supernatant was brought on a Q-sepharose column (500 mL volume), which has been equilibrated with buffer A (32 mM Tris/HCl pH 7.4, 10 mM MgCl2, 1 mM DTE) before. Upon washing with 1.5 column volumes of buffer A a linear salt gradient (0 - 800 mM NaCl in buffer A) was run over an overall volume of 4 L with 4 mL min-1. All steps have been carried out at 4 °C.
Fractions containing the Ras protein were identified by SDS-PAGE following the Lämmli protocol (see section 3.2.1), pooled, concentrated and purified further using a Sephadex G-75 prep grade column equilibrated with buffer B (64 mM Tris/HCl pH 7.4, 10 mM MgCl2, 400 mM NaCl, 2 mM DTE, 0.1 mM DTE). Again SDS-PAGE according to Lämmli (section 3.2.1) allowed for the identification of the fractions containing pure Ras. The corresponding fractions were pooled and concentrated up to about 20 - 50 mg mL-1. Quantification of the protein solution was done by HPLC as described in section 3.2.2. Aliquots of the solution have been frozen in liquid nitrogen and stored at -80 °C.
3.1.2 Expression and Purification of Uniformly
15N-Labeled Ras
Overexpression of uniformly 15N-labeled protein was performed by using T7-vectors in the E. coli strain BL21(DE3). A cell pellet derived from the sedimentation of an overnight culture prepared in LB medium containing carbenicilline with a final concentration of 25 mg L-1 was washed with new minimal medium (NMM, see section 2.6) twice and dissolved in NMM. The cell suspension was transferred into NMM containing 1 g L-1 15NH4Cl as the sole nitrogen source (Neidhardt et al. 1974). Per 1 L main culture
Chapter 3 Methods