BOSQUEJO DE LA ELABORACIÓN DE MODELOS

1

111..11 DDeessiiggnnaannddcclloonniinnggooffddiiffffeerreennttSSRRIIddoommaaiinnvvaarriiaannttss

For following crystallization set-ups DNA fragments comprising varying parts of the SRI domain were amplified by PCR and cloned into the NcoI/NotI sites of a pET24d (G. Stier/EMBL) vector containing an N-terminal His₆-tag (as indicated in chapter 7.2). For NMR spectroscopy the DNA fragment encoding for residues 620 – 719 was amplified by PCR and cloned via its NcoI/Acc65I sites into a modified pET9d vector (G. Stier/EMBL) that contained a TEV protease site directly after the N-terminal z- tag and His₆-tag.

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111..22 PPuurriiffiiccaattiioonnooffSSRRIIddoommaaiinnvvaarriiaannttss

11.2.1 Purification of pET24d expressed SRI domain variants for crystallization

The cell lysate was applied to a His-select nickel column (Quiagen) and each protein eluted in 200 mM imidazole. Fractions containing the desired protein were pooled, diluted in the same volume of MonoS buffer without salt and applied onto a MonoS (Amersham) column. The final purification step comprised a Superdex75 gel filtration and the peak fractions were concentrated for crystallization (Table 10).

Table 10: Purification protocol for crystallization set-ups

Step Buffer composition

Lysis buffer 300 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 % glycerin, 10 mM β-mercaptoethanol, 1/100 proteaseinhibitor mix Wash solution 2 M NaCl

Wash buffer 300 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 % glycerin, 10 mM β-mercaptoethanol, 50 mM imidazole

Elution buffer 300 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 % glycerin, 10 mM β-mercaptoethanol, 200 mM imidazole Mono S 30 mM Mes pH 6.5, 50 mM to 1 M NaCl Superdex75 20 mM Mes pH 6.5, 175 NaCl, 2 mM DTT

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11.2.2 Purification of pET9d expressed SRI domain for NMR spectroscopy

Cell lysates were subjected to affinity chromatography on a first Ni-NTA column (Quiagen), followed by cleavage of the hexahistidine tag with TEV protease and dialysis overnight. The tag and the His₆-tagged protease were removed on a second Ni-NTA column. The pooled fractions were diluted carefully with MonoS buffer (without salt) until the conductivity was around 150 μS/cm. DNA was removed by cation exchange chromatography (MonoS, Amersham). After gel filtration the sample was dissolved in buffer containing 20 mM sodium phosphate pH 6.5, 200 mM NaCl and 2 mM DTT (Table 11). Edman sequencing of the protein confirmed the presence of four additional residues (GAMG) at the N-terminus, which resulted from the cloning strategy.

Table 11: Purification protocol for NMR spectroscopy

Step Buffer composition

Lysis buffer I 300 mM NaCl, 50 mM Tris-HCl pH 8, 5 % glycerin, 10 mM β-mercaptoethanol, 1/100 proteaseinhibitor mix Wash solution I 2 M NaCl

Wash buffer I 300 mM NaCl, 50 mM Tris-HCl pH 8, 5 % glycerin, 10 mM β-mercaptoethanol, 20 mM imidazole Elution buffer I 300 mM NaCl, 50 mM Tris-HCl pH 8, 5 % glycerin,

10 mM β-mercaptoethanol, 200 mM imidazole Dialysis buffer 500 mM NaCl, 50 mM Tris-HCl pH 8, 2 mM DTT Lysis buffer II 500 mM NaCl, 50 mM Tris-HCl pH 8,

10 mM β-mercaptoethanol,

Elution buffer II 500 mM NaCl, 50 mM Tris-HCl pH 8, 30 mM imidazole, 10 mM β-mercaptoethanol,

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111..33 PPhhoosspphhooppeeppttiiddeeiinntteerraaccttiioonnssttuuddiieess

11.3.1 Crystallization set-ups

The phospho-CTD peptides used for binding experiments were chemically synthesized [three-repeat peptide, SPS-YEPTEPS-YEPTEPS-YEPTEPS, E= glutamate mimics phosophoserine (http://www.jerini.com/); two-repeat peptide, SPS- YSPTpSPS-YpSPTpSPS, pS=phosphoserine (http://www.anaspec.com/)]. For crystallization set-ups 1.25-fold molar excess of peptide was co-crystallized with the appropriate SRI domain protein.

11.3.2 NMR-titration

The phospho-CTD peptides used for binding experiments were chemically synthesized [one-repeat peptide, YpSPTpSPS (G. J. Arnold; gene center); two-

repeat peptide, SPS-YpSPTpSPS-YpSPTpSPS, pS=phosphoserine

(http://www.anaspec.com/)]. For NMR titration, increasing amounts of the CTD

peptide were added to a 0.4 mM solution of 15_N,13_{C-labeled SRI domain up to a} 1.25-fold molar excess. Chemical shifts were monitored in 2D 1_H,15_{N HSQC} experiments. The lyophylized, synthetic equivalents of the CTD were dissolved 20 mM sodium phosphate pH 6.5, 200 mM NaCl, 2 mM DTT.

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