Protein Expression and Purification
The gene of full-length human eEF1Bδ was cloned into the pGEX-6P-1 expression vector. The recombinant eEF1Bδ protein containing an N-terminal GST tag was expressed in Escherichia coli BL21 (DE3). Cells were grown at 37 °C, and the protein expression was induced for 3 h with 0.4 mm isopropyl β-d-thiogalactopyranoside when the absorbance at 600 nm reached 0.7–0.8. The cells were harvested by centrifugation at 4800 × g at 4 °C for 30 min. The cell pellets were resuspended in buffer A (20 mm Tris-HCl, pH 7.5, 200 mm NaCl) and stored at −20 °C overnight. After cell lysis by thawing and ultrasonication, the lysate was centrifuged, and the supernatant was applied onto a GST column (GE Healthcare) and washed with buffer B (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, 2.0 mm urea, pH 7.2). The proteins were eluted with buffer C (50 mm Tris-HCl, pH 8.0, 500 mm NaCl) containing 10 mm reduced glutathione and 1 mm dl-dithiothreitol (DTT). After that, the protein was purified by gel filtration chromatography using a Superdex G200 column (GE Healthcare), with buffer C containing 1 m urea to avoid severe aggregation. During the whole purification process, protease inhibitor mixture (Calbiochem) was added according to the manual, and all experiments were carried out at low temperature.
The N-terminal domain (residues 1–153), CAR domain (residues 153–192), catalytic GEF domain (residues 192–281), and a fragment containing the CAR domain plus GEF domain (CAR-GEF region; residues 153–281) of human eEF1Bδ were cloned into a modified pET28a expression vector, with a His-tagged GB1 domain followed by a PreScission protease cleavage site at the N terminus. All eEF1Bδ domains (except the N-terminal domain) were purified by a similar protocol. The proteins were first purified using a Ni2+ column (chelating Sepharose Fast Flow), and then were digested with PreScission protease. The digested product was applied onto the Ni2+ column again. Flow-through solutions containing desired proteins were collected, concentrated, and further purified by gel filtration chromatography using a Superdex 75 column (GE Healthcare). The eEF1Bδ N-terminal domain was expressed mainly in inclusion bodies, and the yield purified from the supernatant was quite low. To purify sufficient eEF1Bδ N-terminal domain, 8 m urea was added into buffer A to resuspend the inclusion bodies. After Ni2+-affinity purification, the eluted fraction was dialyzed to remove urea and imidazole, then concentrated, and further purified by gel filtration. Circular dichroism spectroscopy was used to confirm its refolding (as for proteins purified from the supernatant). The purified protein was collected and digested with 2 mg of PreScission protease, and then a Ni2+ column was used again to separate eEF1Bδ N-terminal domain from GB1 domain.
The human eEF1Bα CAR domain (residues 97–136) and the CAR-GEF region (residues 97–225) were cloned into a modified pET28a expression vector, with a His-tagged SMT3 protein in the N terminus instead of the GB1 domain. These domains were purified by a protocol similar to that in the previous paragraph, except that the His-tagged SMT3 protein was removed by digestion with ULP1 protease.
Wild-type human TCTP was expressed and purified as reported previously (
- Feng Y.
- Liu D.
- Yao H.
- Wang J.
Solution structure and mapping of a very weak calcium-binding site of human translationally controlled tumor protein by NMR.
). The construction of different mutants of the eEF1Bδ CAR domain and TCTP (except the TCTP mutants for paramagnetic relaxation enhancement (PRE) experiments) was carried out by the QuikChange method (
- Hemsley A.
- Arnheim N.
- Toney M.D.
- Cortopassi G.
- Galas D.J.
A simple method for site-directed mutagenesis using the polymerase chain reaction.
). After PCR with mutagenic primers, DpnI was added to digest the methylated nonmutated parental template. The product was transformed into E. coli
TOP10 competent cells. The purification of mutant proteins was similar to that of the wild-type proteins. For the TCTP mutants used in PRE experiments, including C172S, C28S/C172S, C28S/C172S/T116C, C28S/C172S/A127C, and C28S/C172S/D143C, the coding sequence of mutant TCTP was cloned into pET11a or pET30a expression vector without any tag. After expression in E. coli
BL21 (DE3), the cells were resuspended in buffer A without NaCl. After cell lysis and centrifugation, the lysate was loaded onto a DEAE column. The mutant TCTP was eluted with 150 mm
NaCl. The eluate was dialyzed to remove NaCl followed by Q-Sepharose high performance column (GE Healthcare) purification. The mutant TCTP was eluted with a gradient of NaCl concentrations from 50 to 300 mm
. The eluate was concentrated and further purified using a Superdex 75 gel filtration column.
TCTP, eEF1Bα CAR domain, and the CAR-GEF region from fission yeast Schizosaccharomyces pombe and photosynthetic microalga Nannochloropsis oceanica IMET1 were cloned into pET30a for protein expression. The same procedure was used for expression and purification of these two proteins. The plasmid was transformed into E. coli BL21(DE3). Cells were grown at 37 °C, and the protein expression was induced for 5 h with 0.5 mm isopropyl β-d-thiogalactopyranoside when the absorbance at 600 nm reached 1.0. The proteins were first purified using a Ni2+ column (chelating Sepharose Fast Flow) and further purified by gel filtration chromatography using a Superdex 75 column (GE Healthcare). The buffer for gel filtration and final protein storage was 50 mm sodium phosphate buffer, pH 7.0, containing 200 mm KCl, 5 mm DTT, and 5 mm EDTA.
Peptides EDDDIDLFGSDNE, DLFGS, and LFG were synthesized by Sangon Biotech (Shanghai, China). 15N- and 15N-13C-labeled proteins were prepared using the same procedures except cells were grown in M9 minimal media containing 15NH4Cl and [13C]glucose as the sole nitrogen and carbon sources, respectively.
NMR experiments were performed at 298 K on Bruker DMX, AVANCE, and Agilent DD2 600 MHz NMR spectrometers equipped with cryo-probes. All NMR samples contained 0.2–0.8 mm 15N- or 15N/13C-labeled protein in 20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 0.01% 2,2-dimethyl-2-silapentane-5-sulfonate, and 10% (v/v) D2O.
N and 1
C HSQC and three-dimensional CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, HBHA(CO)NH, HBHANH, HCCH-TOCSY, CCH-COSY, and CCH-TOCSY experiments were performed for backbone and side chain assignments of the eEF1Bδ CAR domain in free and TCTP-bound states. Three-dimensional 1
N and 1
C NOESY-HSQC spectra with mixing times of 300 ms were collected to generate distance restraints. All data were processed with FELIX (Accelrys Inc.) or NMRPipe (
- Delaglio F.
- Grzesiek S.
- Vuister G.W.
- Zhu G.
- Pfeifer J.
- Bax A.
NMRPipe: a multidimensional spectral processing system based on UNIX pipes.
) and analyzed with NMRViewJ (
- Johnson B.A.
- Blevins R.A.
NMR View: A computer program for the visualization and analysis of NMR data.
Heteronuclear steady-state 1
N NOE experiments and CLEANEX-PM experiments (
- Hwang T.L.
- van Zijl P.C.
- Mori S.
Accurate quantitation of water-amide proton exchange rates using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a Fast-HSQC (FHSQC) detection scheme.
) were performed using standard pulse programs. Samples of 15
N-labeled human eEF1Bδ CAR-GEF region in free and TCTP-bound states were used in the experiments. The mixing times for CLEANEX-PM experiments ranged from 5 to 500 ms, and the data acquired using short mixing times (5, 10, 15 and 20 ms) were used to estimate the amide-water exchange rates.
Paramagnetic Relaxation Enhancement Experiments
PRE experiments were performed using proteins labeled with 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl methanethiosulfonate (MTSL) (Toronto Research Chemicals, Toronto, Canada) on one free cysteine. Native cysteines on the surface of proteins were mutated to serine to avoid undesired MTSL labeling. The eEF1Bδ CAR domain contains no cysteines, whereas TCTP contains two cysteines, Cys-28p
. (The residues and the mutants of human eEF1Bδ and TCTP are designated by a subscripted suffix δ and p for eEF1Bδ and TCTP, respectively, e.g.
Pro-150 of eEF1Bδ will be represented as Pro-150δ
, and the C172S mutant of TCTP will be represented as C172Sp
.) Because the two cysteines are far away from the binding surface, the C28S/C172S double mutant of TCTP was used in the PRE experiments without interfering with the interaction. Different TCTP or eEF1Bδ cysteine mutants were incubated with MTSL for 16 h at 25 °C in nonreducing buffer, and excess MTSL was removed by dialysis against 20 mm
Tris-HCl buffer, pH 7.5, 200 mm
NaCl for 6 h at 4 °C. Spin-labeled protein was added to other 15
N-labeled protein for NMR experiments. The reduced compound was generated by incubation with 1.5 mm
ascorbic acid for 1 h at 25 °C. The two-dimensional 1
N HSQC spectra of 15
N-labeled proteins were acquired at a 1:1 molar ratio in the oxidized and reduced states (
Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data.
NMR Titration Experiments
The concentration of 15
N-labeled proteins in all titration experiments was 0.1–0.3 mm
. The concentration of stock solution of ligands was 1–5 mm
in the same buffer. All experiments were performed at 25 °C in 20 mm
Tris-HCl buffer, pH 7.5, 200 mm
NaCl, except for the high salt experiment which contained 400 mm
NaCl. The values of chemical shift perturbations (CSP) were calculated using Equation 1
where ΔHN and ΔN are the changes in 1
HN and 15
N chemical shifts, respectively.
The equilibrium dissociation constants (KD
) were estimated by fitting the observed CSPs Equation 2
is the CSP at the theoretical saturated condition obtained from the fit; r is the molar ratio of ligand to protein; Cpro
is the concentration of initial protein solution; and Clig
is the stock concentration of ligand.