3.3.1 Nickel-nitrilotriacetic acid (Ni-NTA) agarose affinity chro-matography
Components of the required buffers can be found in Table 2.6. For purification of sevenfold histidine (His7)-tagged proteins under denaturing conditions, the wash and elution buffers (see 2.6) contained 8 M urea in addition.
Proteins with a N-terminal His7-tag were purified via a Ni-NTA agarose column.
3 ml of resin were used per protein solution from 1 l of expression culture. The resin, equilibrated with wash buffer 1, was incubated with the supernatant from the cell lysis (see 3.2.2) for 1 h at 4◦C under native conditions or at room temperature under denaturing conditions. After washing the column with 10 column volumes of wash buffer 1, bound proteins were eluted by increasing the imidazole concentration in three steps: 1.) 5 column volumes of wash buffer 2, 2.) 5 column volumes of elution buffer 1 and 3.) 5 column volumes of elution buffer 2.
3.3.2 Ion exchange chromatography
Ion exchange chromatography was performed on an ¨AKTA prime low pressure liquid chromatography system with HiTrap columns. Before each run, the HiTrap column was prepared by washing at first with 5 bed volumes of buffer A and then with 5 bed volumes of buffer B. Finally, the column was equilibrated with 5-10 bed volumes of buffer A. Samples were loaded at a flow rate of 1 ml/min onto the equilibrated column. Unbound protein was washed with 5-10 column volumes of buffer A. The elution was performed applying a linear gradient of 0-100 % buffer B at a flow rate of 1-3 ml/min and collecting 2 or 3 ml fractions. Protein elution was detected by measuring the absorption at 280 nm (A280).
3.3.3 Gel filtration
An ¨AKTA basic medium pressure liquid chromatography system was used for gel filtration with a HiLoad Superdex 75 prep grade 16/60 column. 2-5 ml of the con-centrated protein solution containing 10-20 mg protein were loaded onto the column, equilibrated with gel filtration buffer. The gel filtration was performed at a constant flow rate of 1 ml/min. 2 ml fractions were collected.
3.3.4 Reversed phase-high performance liquid chromatography (RP-HPLC)
RP-HPLC systems were operated by Kerstin Overkamp or Gerhard Wolf.
A semi-preparative C18-HPLC column was used for final purification of all CylR2 samples and a semi-preparative C4-HPLC column was used for final purification of all Tir samples. The column was pre-equilibrated with RP-HPLC buffer A (Table2.6).
The elution was performed at a flow rate of 3 ml/min with a linear gradient of 0-100 % RP-HPLC buffer B in 50 min for Tir and in 30 min for CylR2.
3.3.5 Cleavage with TEV-protease
N-terminal His-tags were cleaved with the TEV-protease. At first, His-tagged proteins after affinity chromatography were dialysed at 4 ◦C against two times 5 l TEV-buffer
(see 3.3.1). Then the protein concentration was determined (see 3.4.1) and 0.5-2µg of TEV-protease per 100 mg of protein were added. The cleavage reaction was performed for 16-48 h at room temperature. As the TEV-protease contains a His-tag it was removed via a second affinity chromatography step. After completed cleavage, the protein solution was dialysed against 5 l of wash buffer 1 and applied to a 1-ml Ni-NTA Agarose column. The flow through and the first 5 ml from wash step 1 were collected for further purification.
3.3.6 Concentrating of proteins
Protein solutions were concentrated by ultrafiltration using 0.5, 2 or 20 ml concentra-tors with the appropriate molecular weight cutoff (MWCO). The centrifugation was performed at 4 ◦C as recommended by the supplier (Table2.8). The progress of the ultrafiltration was monitored by measuring the A280.
3.3.7 Protein storage
Proteins in H2O were shock-frozen in liquid nitrogen and dried by lyophilisation.
Lyophilised proteins were stored at -20 ◦C. Proteins in buffer solutions were shock-frozen in liquid nitrogen and stored at -80 ◦C.
3.3.8 Sodium dodecyl sulphate-polyacrylamide gel electrophore-sis (SDS-PAGE)
The discontinuous Tris-glycine buffer system as described by Laemmli [56] was used to separate proteins by molecular weight. The components of the stacking and sep-arating gel are given in Table 3.1. All gels had a separating gel with an acrylamide concentration of 17.5 % and a 3 % acrylamide stacking gel. For detection of proteins with a molecular weight smaller than 8-10 kDa, 2 M Tris/HCl pH 8.8 was used as the separation gel buffer instead of the 1 M buffer [57]. Protein samples were mixed with 4 x protein loading buffer and denatured at 100 ◦C for 5 min. Gels were run at 25 mA and stained with Coomassie blue R-250 by heating for 90 s in the microwave and 10-30 min incubation. For destaining, several 90 s heating steps in the microwave
were performed and gels were completely destained by shaking overnight. Finally, the SDS-gels were imaged and digitized for documentation.
Table 3.1: Composition of the 17.5 % SDS-gels
Seperating gel Stacking gel
Rotiphorese Gel 30 3 ml 250µl
1 M or 2 M Tris/HCl pH 8.8 1.88 ml
-1 M Tris/HCl pH 6.8 - 313µl
H2O 30 µl 1.88 ml
10 % SDS 50 µl 25µl
TEMED 2.5 µl 2µl
10 % APS 50 µl 25µl
3.3.9 Edman degradation
N-terminal protein sequencing was performed via Edman degradation [58] by U. Pleß-mann in the group of Prof. Dr. Klaus Weber.
3.3.10 Limited proteolysis
Limited proteolysis was used to identify the rigid core of Map in complex with CesT [59]. Proteolysis was performed with the proteases trypsin, proteaseK, elastase and chymotrypsin. A∼30µM protein solution in 20 mM HEPES pH 7.0 and 150 mM NaCl was digested at protein:protease ratios of 50:1, 100:1, 1000:1 and 10000:1 at room temperature. The reaction was stopped after 5, 10, 20, 30, 60 and 120 min by addition of SDS-PAGE loading buffer and boiling for∼5 min at 100 ◦C. All samples were analysed by SDS-PAGE.
3.3.11 Peptide synthesis
Solid-phase peptide synthesis (SPPS) was performed by Kerstin Overkamp. Raw peptides were purified via RP-HPLC.
3.3.12 Introduction of a paramagnetic center
3.3.12.1 Site-directed spin-labeling (SDSL)
SDSL is a technique to introduce a spin label into a protein based on a specific reac-tion between the spin label and an amino acid. In the most commonly used proce-dure, a unique sulfhydryl group is selectively modified with a nitroxide reagent [60].
Single cysteine mutants for the attachment of a spin label were generated by site-directed mutagenesis (see 3.1.5) and modified with the thiol-specific nitroxide spin label reagent (1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate (MTSL). 100µg/µl MTSL in cold acetone was added with a 3 to 5-fold molar excess and incubated overnight at room temperature. Unreacted spin label was removed by size exclusion chromatography via a PD-10 column. Complete labeling with MTSL was verified by ESI-MS (see 3.4.3).
3.3.12.2 Copper-binding
Introduction of paramagnetic Cu2+ was achieved by prepending the amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif to the N-terminus of CylR2 via recombi-nant cloning. Cu2+ was bound by adding a 3 to 5-fold excess of CuCl2 dissolved in H2O. Excessive Cu2+ was removed by dialysis against MOPS buffer. MOPS buffer was used to minimize the interaction between buffer molecules and Cu2+ [61].