6. Materials and Methods
6.3 Biomolecular Methods
6.3.15 DEER Experiments
DEER experiments were performed with glassy solutions of proteins (protein buffer + 20
% glycerol, v/v) at cryogenic temperatures (50 ° K) in Q-Band, using Elexsys E580 spectrometer Bruker Biospin equipped with a helium gas flow system CF935 Oxford Instruments. Singly labeled protein solutions were used as background correction. DEER experiments were performed by Julia Borbas, Christian Hintze and Artem Feedosev (AG Drescher).
101 6.3.16 Stability Measurements in E. coli Lysates
A single colony of E. coli GH371 was picked to inoculate 5 ml LB-medium. The culture was incubated at 37 °C and 180 rpm shaking overnight. The OD600nm of the culture was adjusted to 2.5 with LB-medium. 1.5 ml of this culture (approx. 3.75x109 cells) were pre-incubated at the designated temperature for 10 minutes, before being pelleted by centrifugation (3320 x g, 10 minutes). The supernatant was removed and the pellet was lyzed with 75 µl B-Per lysis reagent. The cell suspension (59.4 µl) was transferred to a new tube and mixed with 10 mM 6 (0.6 µl), resulting in 100 µM 6. The CW-EPR spectra were recorded over time at the designated temperatures. Experiments were conducted as triplicates and the half-life of 6 was calculated.
6.3.17 Stability Measurements in E. coli Culture
A single colony of E. coli GH371 was picked to inoculate 5 ml LB-medium. The culture was incubated at 37 °C and 180 rpm shaking overnight. The OD600nm of the culture was adjusted to 2.5 with LB-medium. The cell suspension (59.4 µl) was transferred to a new tube and mixed with 10 mM 6 (0.6 µl), resulting in 100 µM 6. The CW-EPR spectra were recorded over time at the designated temperatures. Experiments were conducted as triplicates and the half-life of 6 was calculated.
6.3.18 Expression and Purification of GFP Mutants Containing ncAA 3
E. coli Topten, GH371 or JX33 [195] cells were co-transformed with a pEVOL-based plasmid (encoding a PylRS gene under a constitutive glnS’ promotor and a PylRS gene under an araBAD promotor and tRNAPyl under a proK promotor) and a pBAD_GFP plasmid (e.g. pBAD_Flag-GFP-Y39TAG-6His [187]: encoding a GFP gene with a C-terminal his6tag and an N-C-terminal flag-tag and an amber codon at Y39 under control of an araBAD promotor). A single colony was picked from an LB-agar plate and was used to inoculate 5 ml LB medium supplemented with 50 µg/ml carbenicilin and 34 µg/ml chloramphenicol (for plasmid propagation) and was incubated at 37 °C, 200 rpm overnight. 100 µl of this overnight culture was diluted in fresh 5 ml LB medium supplemented with 50 µg/ml carbenicilin, 34 µg/ml chloramphenicol, 1 – 2 mM ncAA 3 and was incubated at 37 °C, 200 rpm until the OD600 nm reached 0.4 – 0.5. The expression was induced by adding 0.02 % (w/v) L-arabinose into the medium. The culture was further incubated at 37 °C, 200 rpm for 4 h and was harvested by centrifugation (3320 x g, 10 min). The supernatant was discarded and the pellets were stored at -20 °C.
The pellets were lyzed at room temperature with 0.5 ml B-Per lysis buffer (Thermo Scientific) supplemented with 1x Complete (-EDTA) protease inhibitor cocktail (Roche) for 30 minutes at 1400 rpm (Thermomixer). Cell debris was removed by centrifugation
102
(20817 x g, 5 min) and the supernatant was incubated with 10 mM imidazole and 50 µl Ni-NTA resin (corresponding to max. binding of 250 – 500 µg protein). The suspension was incubated at r.t. with 500 rpm shaking for 30 minutes and was then filtered through a filter spin column (Pierce) by centrifugation (1486 x g, 10 s). The Ni beads were washed with 4 x PBS (2 x 700 µl), wash buffer I (3 x 700 µl; 50 mM NaH2PO4, 300 mM NaCl, pH
= 8.0, 20 mM imidazole) and once with wash buffer II (1 x 700 µl; 50 mM NaH2PO4, 300 mM NaCl, pH = 8.0, 50 mM imidazole), by resuspending the beads centrifugation (1486 x g, 10 s). To elute the proteins, the Ni beads were incubated with elution buffer (2 x 50 µl; 50 mM NaH2PO4, 300 mM NaCl, pH = 8.0, 500 mM imidazole) for 10 minutes at r.t.
and the protein was collected by centrifugation (1486 x g, 10 s). The eluted protein fractions were analyzed using SDS-PAGE and mass spectrometry and were quantified using a BCA Kit (Thermo Scientific).
6.3.19 Expression and Purification of GFP Mutants Containing ncAA 6
Expression with ncAA 6 was performed according to 6.3.18 with minor changes. 1 - 3 mM of ncAA 6 were added upon induction of the expression with 0.2 % (w/v) L-arabinose. The expression was performed between 4 – 16 h and the expression culture was harvested by centrifugation at 4 °C and the pellets were immediately shock-frozen in liquid nitrogen and stored at -20 °C. The lysis and purification steps were performed on ice, with pre-cooled reagents. 1 mM PMSF was used instead of Complete (-EDTA) protease inhibitor cocktail (Roche) during lysis. The lysis was performed for 10 minutes and the Ni-NTA binding step for 10 minutes on ice. The eluted protein fractions were analyzed using SDS-PAGE, mass spectrometry and were quantified using a BCA Kit (Thermo Scientific). The integritiy degree was measured by CW-EPR.
6.3.20 Measurement of Cellular GFP Fluorescence
5 ml GFP expression cultures were pelleted by centrifugation (3320 x g, 10 min), the supernatant was discarded and the pellets were resuspended in 1 x PBS (5 ml). This washing step was repeated once. The pellets were finally resuspended in 5 ml PBS (1x) and cellular GFP fluorescence was determined using a Tecon M200 plate reader (excitation wavelength = 475 nm; emission wavelength = 510 nm). The fluorescence was normalized to the cell number (optical density at 600 nm).
6.3.21 Construction of pBAD_TRX-His6 Expression Vectors
pBAD_TRX-His6 (pSuE177) was constructed by Susanne Ermert during her bachelor thesis. Briefly, E. coli thioredoxin (with an N-terminal S-tag and a C-terminal Enteropeptidase site) was amplified by PCR from pET32a-TRXtag (Addgene). The PCR forward primer was designed to exhibit a 5’ NcoI site and the revesie primer a NotI sites
103 as well as a His6tag. The digested PCR fragment was cloned into pBAD_GFP_Y39TAG.
Cloning was verified by sequencing.
6.3.22 Expression and Purification of TRX Mutants Containing ncAA 3 The expression of E. coli thioredoxin mutants was performed analogous to 6.3.18.
6.3.23 Expression and Purification of TRX Mutants Containing ncAA 6 The expression of E. coli thioredoxin mutants was performed analogous to 6.3.19.
6.3.24 Construction of pBAD_Tat-GFP-His6 Expression Vectors
The Tat-derived arginine rich motif was cloned as an N-terminal GFP fusion into progenitor vector pBAD_GFP-wt (pDaS 72), replacing the N-terminal flag-tag with NheI and NcoI restriction sites and a GGSAS-linker region. The Tat peptides were created by primer extension reactions using template primers listed in 8.2. The primer was extended using Klenow fragment DNA polymerase, before the extension product was purified, digested with NheI and NcoI and ligated into the final vector (e.g. a pBAD_TAT-GFP-His6 encoding an N-terminal Tat-derived ARM peptide fused to GFP with a C-terminal his6tag under control of an araBAD promotor).
6.3.25 Expression and Purification of Tat_GFP Mutants Containing ncAA 3
E. coli BL21(DE3) gold cellsii were co-transformed with pEVOL_PylRS_AF (pDaS70) and a pBAD_Tat-GFP-His6 plasmid. A single colony was picked from an LB-agar plate and used to inoculate 5 ml LB medium supplemented with 50 µg/ml carbenicilin and 34 µg/ml chloramphenicol (for plasmid propagation) and the culture was incubated at 37 °C, 200 rpm overnight. 1 ml of this overnight culture was diluted into 50 ml LB medium supplemented with 50 µg/ml carbenicilin, 34 µg/ml chloramphenicol and 2 mM ncAA 3 and was incubated at 37 °C, 200 rpm until the OD600 nm reached 0.5 – 0.6. The expression was induced by adding 0.04 % (w/v) L-arabinose into the medium and the culture was incubated at 37 °C, 200 rpm for 6 h. The cell suspension was harvested by centrifugation (3320 x g, 10 min) and the supernatant was discarded and the pellets were stored at -20 °C.
The pellets were thawed and lyzed at room temperature with 2 ml B-Per lysis buffer (Thermo Scientific) supplemented with 1 mM PMSF for 30 minutes at 1000 rpm (Thermomixer). The suspension was centrifuged (5 min, 20817 x g, room temperature), the supernatant was discarded and the pellet was extracted with 2 ml Buffer-Z (8 M urea, 0.1 M NaCl, 20 mM HEPES, pH = 8) for 3 h at 1000 rpm at room temperature. The suspension was centrifuged (30 min, 20817 x g, room temperature) and the supernatant
ii N-terminal Tat-GFP proteins were found to be partially digested using E. coli strain non-deficient of Lon and OmpT proteases
104
was incubated with 100 µl Ni-NTA slurry (corresponding to max. binding of 500 – 1000 µg protein) at r.t. at 500 rpm for 30 minutes. The suspension was filtered using a filter spin column (Pierce) by centrifugation (1486 x g, 10 s) and the Ni-beads were washed with Buffer-Z (1 x 500 µl), four times (500 µl) with wash Buffer-Z wash buffer I (Buffer-Z containing 20 mM imidazole) and once with 500 µl Buffer-Z wash buffer II (Buffer-Z containing 50 mM imidazole). The beads were resuspended and filtered by centrifugation (1486 x g, 10 s). To elute the proteins, the Ni-beads were incubated with Buffer-Z elution buffer (2 x 50 µl; Buffer-Z containing 500 mM imidazole) for 10 minutes at r.t. and the protein was collected by centrifugation (1486 x g, 10 s). The eluted protein fractions were analyzed using SDS-PAGE and mass spectrometry and were quantified using a BCA Kit (Thermo Scientific). The proteins were refolded by stepwise dialysis against 20 mM Tris-HCl, pH = 8, 10 % (v/v) glycerol with decreasing urea concentrations.
6.3.26 5’-end Labeling of TAR RNA with γ-32P-ATP
To radioactive label the 5’-end of TAR RNA, T4 polynucleotide kinase (PNK) was employed. The γ-32phosphate of γ-32P-ATP was transferred to the 5’-OH terminus of the RNA oligonucleotide.
For labelling the following protocol was applied:
Component Volume [µl] Concentration
Nuclease-free water 42 -
TAR RNA (10 µM) 1 10 pmol
γ-32P-ATP (10 µCi/µl) 1 0.2 µCi
T4-PNK buffer (10x) Buffer A 5 1x
T4-PNK 1 10 U
The reaction mixture was incubated at 37 °C for 1 h, before being quenched with 1.5 µl EDTA (0.5 M). The solution was heated to 75 °C for 10 minutes to denature T4-PNK.
The reaction mixture was allowed to cool to r.t. and was purified using a GE Healthcare G-25 column. The obtained labeled RNA solution (50 µl) was diluted with 20 µl unlabeled RNA (10 µM), resulting in a 3 µM solution.
6.3.27 Electromobility Shift Assays of Tat-GFP Proteins and TAR-RNA
The EMSA binding assay was performed at 4 °C in a cold room. 5’-32P-labelled TAR RNA (10 nM) was incubated with decreasing concentrations of Tat-GFP proteins in 50 mM Tris-HCl pH = 8, 50 mM KCl, 0.05 % Triton-X and 5 % (v/v) glycerole for 30 min at 4
°C. A 12 % native PAGE gel (0.5 mm, 50 cm length) was pre-run in 1x TB for 60 minutes at 15 W and 4 °C. After incubation, the reaction mixture was loaded onto the native PAGE gel and was run at 15 W and 4 °C for 60 minutes. The gel was transferred to Whatman paper and covered with plastic foil. The gel was dried in a gel dryer (80°C for
105 60 minutes) prior to exposure to a phosphor imager screen (Fuji) overnight. The imager screen was analyzed on a phosphorimager (BioRad) and the data was processed using Quantity one (BioRad).
6.3.28 Crosslinking Experiments of Tat-GFP Proteins and TAR-RNA
Crosslinking experiments were conducted with 10 nM 5´-32P-labelled TAR RNA and 2 µM of Tat-GFP proteins in binding buffer (50 mM Tris-HCl pH = 8, 50 mM KCl, 0.05 % Triton-X and 5 % (v/v) glycerole) supplemented with 16 µM methylene blue (8 eq.). The RNA-protein binding mixture was incubated at 4 °C for 30 minutes before being irradiated in an open reaction tube (1.5 ml) for 2 minutes at 900 rpm shaking and 4 °C with red light (using an Euromex cold light illuminator equipped with a red R60 type filter;
light-intensity: 9). A 10 % denaturing PAGE gel (0.5 mm, 50 cm length) was pre-run at 2300 V for 60 minutes in 1 x TBE buffer to warm up to approx. 50 °C. The reaction mixture was quenched with formamide buffer and loaded onto the PAGE gel. The gel was run at 2300 V for 30 minutes in 1 x TBE buffer, was transferred to Whatman paper and covered with plastic foil. The gel was dried in a gel dryer (80°C for 60 minutes) prior to exposure to a phosphor imager screen (Fuji) overnight. The imager screen was analyzed on a phosphorimager (BioRad) and crosslinking effects were quantified using Quantity one (BioRad).
Additional crosslinking experiments for Tat-R49→3 and Tat-ins47→3 were performed with 10 nM 5´-32P-labelled TAR RNA and 1 µM of Tat-GFP proteins. The samples were incubated with 8 eq. of methylene blue and were irradiated as above for 10 minutes before being analyzed on a 10 % denaturing PAGE gel.
6.3.29 SDS-PAGE Electrophoresis
Protein samples were mixed with SDS loading buffer and were incubated at 95 °C for 5 minutes for denaturation. The samples were allowed to cool to room temperature and were loaded on an SDS Gel with broad range protein marker (NEB) as a reference. SDS gels were run for 1 h 15 minutes at 120 V. After electrophoresis, the gels were washed with water and stained in Comassie (Carl Roth) staining solution for 1 h with shaking.
The gels were developed in destaining solution for 2 h and finally incubated in water to remove residual background staining.
6.3.30 Determination of Protein Concentrations using a BCA Assay
Protein concentrations were measured using a BCA Kit (Thermo Scientific) following the manufacturers’ protocol.
106
6.3.31 Protein Mass Spectrometry
For molecular weight determination using the LTQ-Orbitrap, protein samples were dialyzed against water using Slida-A-Lyzer dialysis units (MWCO: 10 kDa) (Protein spectra were deconvoluted with ProMassDeconvolution V2.5 software).
For Tryptic digests and subsequent tandem mass spectrometry (LC-MS/MS) analysis on a LTQ-Orbitrap mass spectrometer (Thermo Fisher) and an Eksigent nano-HPLC, SDS-gel protein bands were excised with a scalpel and processed by the proteomics facility.
The reversed-phase LC column was 5 μm, 200 Å pore size C18 resin in a 75 μm i.d. × 10 cm long piece of fused silica capillary (Hypersil Gold C18, New Objective). The reversed-phase LC column was washed ith 95% mobile reversed-phase A (0.1% formic acid) and 5%
mobile phase B (0.1% formic acid in acetonitrile). Peptides were eluted using a linear gradient of 5 % mobile phase B to 40 % in 65 min, then to 80 % B in an additional 5 min, at 300 nL/min.
The mass spectrometer (LTQ-Orbitrap) was operated in a mode, in which each full MS scan (30 000 resolving power) was followed by five MS/MS scans. The five most abundant molecular ions were selected and fragmented by collision-induced dissociation (CID). The collision energy was set to 35% in the LTQ ion trap with dynamic exclusion.
LC-MS/MS (tandem mass spectra) were recorded and analyzed with a homemade protein database using Mascot (Matrix Science). Cysteine alkylation by iodoacetamide and methionine oxidation was considered.
107
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