Expression and purification of full-length wild-type eIF4E and the K119A mutant
The plasmid containing the full-length cDNA for human eIF4E (gift from Dr N. Shimma, Chugai
Pharmaceutical Co., Japan) was expressed in Escherichia coli BL21 (DE3) (Miura et al., 2003). The
eIF4E K119A mutant was generated by PCR-directed mutagenesis and cloned into similar E. coli
expression vectors as the wild-type protein and sequenced. Both proteins were grown at 37ºC in either
Luria Bertani (LB) or M9 minimal medium containing ampicillin (100 g/L), and using freshly
transformed cells (BL21 DE3). Each liter of minimum M9 media was supplemented with 1g of
[15N]ammonium chloride (Cambridge Isotopes Laboratory, Andover, MA), 2g of [ 13C]glucose (or 4g of
[12C]glucose), 2 ml of 1M MgSO4, 1 ml of 0.1M CaCl2, 10 ml of BME vitamins solution and trace
elements (citric acid-H2O, FeCl3-6H2O, ZnCl2-4H2O, MnCl2-4H2O, CoCl2-6H2O, CaCl2, CuCl2-2H2O,
H3BO3, Na2MoO4-2H2O, NiSO4-6H2O). Protein expression was induced at 30 ºC for 18 h by addition of
0.5 mM isopropyl--D-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation (7,000
rpm, 15 min., 4°C) and frozen at -20°C. The cell pellet was then resuspended in 30 ml of lysis buffer (50
mM phosphate buffer pH 7.4, 100 mM NaCl, 0.5 mM EDTA, 5 mM dithio-1,4-threitol (DTT), 150 l
IGEPAL-CA 630, 5 mM -mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mg/ml
lysozyme) and cells were disrupted by sonication on ice (four 10 seconds pulses with 30 seconds
pauses). The lysate was cleared by centrifugation (18,000 rpm, 30 min., 4°C) and the supernatant was
clarified by filtration on a 0.45 m membrane and loaded onto m7GDP-sepharose (cap column). After
four washes (30 ml each; 1. lysis buffer, 2. wash buffer [50 mM phosphate buffer pH 7.4, 100 mM
NaCl], 3. wash buffer containing 0.1 mM GTP and 4. wash buffer), protein was eluted with ~ 60 ml of
elution buffer (wash buffer containing 8 mM m7G and 2 mM DTT, or 2 M NaCl). The eluate was
concentrated using 15 ml amicon ultra centrifugal filter device (5 K MWCO, Millipore) to 10 ml, and
extensively dialyzed in 50 mM phosphate buffer (pH 7.4), 100 mM NaCl and 2 mM DTT. Salt
concentration was decreased to 25 mM and sample was applied to an anion-exchange (Mono-Q,
Amersham Biosciences). The protein was eluted with a salt gradient (25-1000 mM NaCl) in 50 mM
phosphate buffer pH 7.4.
Isotopically enriched eIF4E was prepared from cells grown on minimal M9 media containing
[ N]ammonium chloride with or without [13C6]glucose (Cambridge Isotopes Laboratory, Andover,
MA). The uniformly 15N-2H labeled eIF4E was prepared as described (Gardner and Kay, 1998).
The protein concentration was measured at 280 nm using an extinction coefficient of 53440 M-1 cm-
. The final yield of soluble protein was ~ 10 mg, ~ 6 mg or ~ 2 mg /L of bacterial culture grown in
either LB medium, M9 minimum medium or M9 minimum D2O medium, respectively.
NMR spectroscopy. NMR experiments for backbone assignments were acquired at 600 MHz on a
Varian INOVA spectrometer equipped with an HCN probe and triple axis gradients at 20ºC. Due to the
low concentration of eIF4E and time-dependant stability we employed non-uniform sampling versions
of the HNCA and HNCO experiments (Rovnyak et al., 2004) to accelerate the acquisition time. For both
experiments a total of 350 complex fids were accumulated using a sampling schedule generated using
the Coast software corresponding to a maximum of 60 and 32 points in the 13C and 15N dimensions,
respectively. A total of 256 scans were acquired per increment, yielding a total acquisition time of about
5 days. Non-uniform sampling spectra were processed using maximum entropy reconstruction using the
Rowland NMR toolkit (RNMRTK; http://www.rowland.org/rnmrtk) and converted to NMRPipe
(Delaglio et al., 1995) and Sparky formats (Goddard and Kneller, 2003). Typically, sampled
experiments were processed with NMRPipe and analyzed with Sparky.
Heteronuclear 1H-15N NOE spectra were recorded in an interleaved manner in which each
sequential fid was recorded with and without 4 seconds of presaturation plus a 1 second recycle delay
using the pulse sequence described by Farrow et al. (1994). Errors for the heteronuclear NOE values
were estimated from the root mean square variation of noise in empty regions of the two spectra as
described previously (Dutta et al., 2004). Translational diffusion coefficients were obtained using an 15N
isotope-filtered sequence described by Nesmelova et al. (2004).
Structure calculation. Distance restraints were obtained from 3D 15N-edited and 13C-edited
NOESY spectra (100 ms mixing time) and additional longer-range NH-NH distance restraints were
obtained from a 200 ms mixing time 3D 15N-edited NOESY spectrum acquired on a uniformly 15N-2H
labeled eIF4E sample to help overcome the effects of spin-diffusion. Manually assigned distance
restraints were classified according to the peak intensities. Also, a lyophilized sample of 15N-labeled
eIF4E was dissolved in D2O, and 1H-15N HSQC experiments were recorded at 30 min intervals at 293
K. Amide protons were considered to exchange and thus to be involved in hydrogen bonding if they
were still visible in the second HSQC. For each hydrogen bond, two distance restraints were applied for
HN(i)-O(j) and N(i)-O(j).
Structures were calculated using the CNS protocol (Brunger et al., 1998) starting with an extended
molecule. After the first round, the resulting models were used to identify hydrogen bonds. Eighty were
consistently observed. After another round, the structure having the lowest energy and least violations
was used as input for the TALOS program (Cornilescu et al., 1999). This calculation with TALOS
generated 276 and dihedral angle restraints that were used in a final structure calculation.
The quality of structures obtained was assessed with PROCHECK (Laskowski et al., 1993)
without the unstructured residues 1 to 35. The graphic representations of the three-dimensional
structures were performed using MOLMOL (Koradi et al., 1996). Coordinates were deposited in the
Protein Data Bank under PDB ID code 2GPQ.
Fluorescence spectroscopy. Cap titration and fluorescence measurements were carried out as
described previously (Kentsis et al., 2001). Briefly, 2 M protein was incubated with the increasing
concentrations of m7GDP, m7GTP or m7GpppG (0-100 M) in 50 mM NaH2PO4 pH 7.4, 100 mM NaCl,
1 mM DTT. Fluorescence measurements were performed in a 0.3 x 0.3 cm2 fluorescence cuvette
(Hellma, Forest Hills, NY), using Jasco FP6500 fluorimeter. Samples were excited at 295 nm with 3 nm
bandwidth and fluorescence of tryptophans was selectively monitored in the course of the titrations
between 300 and 450 nm. Collected emission spectra were integrated between 300 and 450 nm, and
spectral contribution from eIF4E protein was determined by subtraction of intrinsic fluorescence of the
different cap analogues as determined from identical titrations in the absence of protein. Since the
reduction of the excitation intensity by the cap analogues (i.e. inner filter effect) leads to an inaccurate
estimation of the association constants (Wieczorek et al., 1998), the resulting integrals were further
corrected for this effect using extinction coefficient of 3207, 3202 and 6255 M-1.cm-1 at 295 nm for
m7GDP, m7GTP and m7GpppG, respectively (Lakowicz, 1999). Binding isotherms were fit according to
a heuristic single site binding expression (see supplementary figure 2).
Circular dichroism spectroscopy. Far-UV CD spectra were collected using Jasco-810
spectropolarimeter with 0.847 cm tandem cuvette (Hellma) at room temperature as reported previously
(Kentsis et al., 2001). The solution conditions were 50 mM phosphate buffer (pH 7.4), 100 mM NaCl, 1
mM DTT. All of the measurements were repeated two times with a 1 nm bandwidth. Relative ellipticity
was converted to mean residue molar ellipticity according to Fasman (1996).
Mass spectroscopy. Purified apo-eIF4E was extensively dialyzed against 20mM NH4OAc buffer
(pH 6.5) and then lyophilized. An aliquot of the apo-protein was solubilized in a MS buffer (1:1
acetonitrile/water solution with 0.1% trifluoroacetic acid) to a final concentration of 40 M. Ligand-
bound eIF4E was prepared by incubating 0.6 mM of m7GDP with 0.12 mM (5:1 ligand to protein) in the
MS buffer for 1 minute at room temperature. Both samples were electrosprayed directly into a PE Sciex
API-III triple quadrapole mass spectrometer. Experimental masses were calculated by Hypermass and
Hypermass Reconstruct programs within Biomultiview 1.0 (supplied by the instrument’s manufacturer).
Note that association constants for cap cannot be calculated from this data due to difference in ionization
efficiencies of apo and cap-bound eIF4E.
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