Eichmann et al. 10.1073/pnas.0914300107
SI Text
Material and Methods.Expression and labeling of RNCs.Plasmids en-coding the various nascent chain constructs were generated using standard cloning procedures and transformed into the Escheri-chia colistrain BL21(DE3)Δtig::kanfor expression. The basic protocol was adapted from (1). In brief, cells were grown in 4 l TB supplemented with100μg∕ml ampicillin at 37 °C while shak-ing at 140 rpm. At optical cell densities (OD600) of 0.8, cells were pelleted by centrifugation at5;000 ×gfor 30 min at 4 °C, washed in M9 salt solution and sedimented again. For isotopic labeling, the cell pellets were resuspended in 1.7 l of prewarmed minimal (M9) medium (D-glucose and/or NH4Cl were substituted for13C D-glucose and15NH4Cl, respectively) containing100μg∕ml am-picillin and vitamins for basal medium eagle (MP). After 1 h in-cubation at 37 °C at 140 rpm, expression of RNCs was induced by the addition of 1 mM isopropylthioβ-D-1-galactopyranoside (IPTG). Cells were grown for 2.5 h in presence of IPTG prior to pelleting for 30 min at5;000 ×gat 4 °C and stored at−80°C.
Purification of RNCs.StrepII-tagged RNCs were prepared as de-scribed (2) with the following modifications: Frozen cell pellets from labeled cultures were thawed on ice, resuspended in buffer I (50 mM HEPES-KOH pH 7.4, 100 mM potassium acetate, 15 mM magnesium acetate, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF), protease inhibitor cock-tail (Roche)) and lysed by French Press. After centrifugation for 30 min at30;000×g, the supernatant was loaded onto a high-salt sucrose cushion (30%w∕v sucrose in 50 mM HEPES-KOH pH 7.4, 1 M potassium acetate, 15 mM magnesium acetate, 1 mM DTT, 0.5 mM PMSF) for sedimentation of crude ribosomes at 200;000 ×gfor 4 h at 4 °C. The pellets containing RNCs were resuspended on ice in buffer I containing 5 mM ATP overnight or for 2 h by shaking at 210 rpm. The RNCs were bound to Strep-Tactin sepharose (IBA) for 30 min while slowly rotating at 4 °C.
Afterward the matrix was transferred to a column, washed with 3 column volumes (CV) ST-buffer (50 mM HEPES-KOH pH 7.4, 100 mM potassium acetate, 15 mM magnesium acetate, 1 mM DTT) containing 5 mM ATP, 3 CV hs-buffer (50 mM HEPES-KOH pH 7.4, 0.8 M potassium acetate, 15 mM magnesium ace-tate, 1 mM DTT) and 4 CV ST-buffer. The 3xStrep-Smt3 tag was removed by on-column digestion with Ulp1 protease for 6 h at 4 ° C in 1.5 CV R-buffer (30 mM HEPES-KOH pH 7.4, 50 mM po-tassium acetate, 15 mM magnesium acetate, 1 mM DTT). After-wards, the flow-through containing the released RNCs was passed over a second Strep-Tactin column to remove uncleaved RNCs. Free RNCs were collected by centrifugation for 40 min at 200;000 ×gat 4 °C. The pellet was washed and resuspended within 2 h on ice in SP-buffer (30 mM dTris-HCl pH 6.8, 50 mM KCl, 12 mM MgCl2, 2 mM DTT) shaking at 210 rpm, centrifuged again for 40 min at180;000×gand resuspended in∼300μl SP-buffer. RNCs were frozen in liquid nitrogen and stored at−80°C or applied directly to NMR. For biochemical analysis, RNCs were eluted from the Strep-Tactin matrix with EB (50 mM HEPES-KOH pH 7.4, 100 mM potassium acetate, 15 mM magnesium acetate, 1 mM DTT, 1 mM PMSF) containing 2.5 mM D-desthiobiotin (IBA). RNCs were incubated 30 min at 37 °C prior to Ulp1 treatment. The digested RNCs were centri-fuged and the RNCs were resuspended in EB. Samples of the
Preparation of unlabeled and labeled E. coli ribosomes.BL21(DE3) Δtig::kancells were grown under the same conditions as for the expression of RNCs in medium containing40μg∕ml kanamycin.
Ribosomes were prepared as described above, except that affinity chromatography was omitted. After the first ultracentrifugation step, the resuspended ribosomes were loaded directly onto a sec-ond high-salt sucrose cushion supplemented with 5 mM ATP and pellets were washed and resuspended in SP-buffer as described (see above).
Preparation of SH3-T1-F and SH3-T2-F.As control for SH3-T1-RNCs and SH3-T2-RNCs,15N-labeled SH3-T1-F and SH3-T2-F were added to ribosome samples following the procedure of Hsu et al. (3). The truncated SH3 versions (T1 and T2) were cloned on the same basic vector as the nascent chain constructs but lack-ing the SecM-linker-TEV elements. Expression was performed in BL21(DE3)Δtig::kancells. Cells were grown as described above in 2 l TB to an OD600of 0.8. For isotopic labeling, cells were re-suspended in 1.3 l M9 medium, grown for 1 h at 17 °C prior to induction with 0.1 mM IPTG and incubated for 12 h at 17 °C.
Cells were lysed according to the RNC purification protocol (see above) and the lysate was subjected to Strep-Tactin affinity chromatography. After the binding of the truncated SH3 ver-sions, the column was washed with ST-buffer, ST-buffer contain-ing 5 mM ATP, hs-buffer and again with ST-buffer. For buffer exchange, the column was equilibrated in D-buffer (10 mM HEPES-KOH pH 7.4, 50 mM KCl, 6 mM MgCl2, 1 mM DTT) and washed with F-buffer (10 mM dTris-HCl pH 6.8, 50 mM KCl, 6 mM MgCl2, 1 mM DTT). Ulp1 digestion was performed in C-buffer (20 mM dTris-HCl pH 6.8, 50 mM KCl, 6 mM MgCl2, 1 mM DTT) and released SH3 versions were supplemented with protease inhibitor cocktail and concentrated in a 1 kDa MWCO dialysis tube on absorbent material (Spectra/Gel) at 4 °C. After-wards, the proteins were dialyzed overnight against C-buffer con-taining protease inhibitor cocktail, aggregates were removed by centrifugation and the samples were measured directly by NMR.
Expression and purification of nonribosomal SH3/m10 constructs.The SH3-F and SH3-m10-F domain constructs were generated from the SH3-/SH3-m10-RNC vectors by introduction of a stop codon after the SH3/m10 coding sequences, respectively. The SH3-SecM-F construct was released from ribosomes by mutating the critical residue R163 (150FxxxxWIxxxxGIRAGP166) to alanine in the SecM coding sequence on the SH3-RNC plasmid. Expression and purification of the constructs was performed as described for the truncated SH3 versions. Cells were grown to an OD600of 0.8 in 1.5 l TB and then transferred to 1 l M9 medium for labeling. The cells were harvested 6 h after induction, lysed and the proteins were bound to Strep-Tactin matrix. After the wash steps, the col-umn was equilibrated with D2-buffer (20 mM Tris-HCl pH 6.8, 50 mM KCl, 6 mM MgCl2, 1 mM DTT) for Ulp1 cleavage. The samples were passed over a second Strep-Tactin column, frozen in liquid nitrogen and stored at−80°C.
NMR Spectroscopy and analysis. Sequential assignment was performed with the standard protocol for13C;15N-labeled sam-ples applied to15N;13C-labeled SH3-F, SH3-m10-F, SH3-T1-F and SH3-T2-F free in solution at concentrations of 0.1 mM
CARA (10).
Almost complete sequential backbone assignment of SH3-F, SH3-m10-F, and side-chain assignment was obtained for SH3-F. The sequential assignment of SH3-T1-F and SH3-T2-F is less complete because of peak overlap in particular on the side-chain resonances. Nevertheless, 78% of the backbone reso-nances could be determined for both SH3-T1-F and SH3-T2-F, and 66% and 53% for the side-chain resonances, respectively.
each case. For this purpose spectra were used for which the line widths of the cross peaks were not determined by the apodization function.
15N-edited and14N-edited diffusion measurements with varied diffusion coding gradient strengths (4 ms total duration) were re-corded by using a diffusion delay of 100 ms. A total 2048 tran-sients were measured per gradient strength.
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Fig. S1. Analysis of purified SH3-RNCs. RNCs were applied to SDS–PAGE and ribosomal proteins were visualized by Coomassie staining (A) and the nascent chains (NC) detected via the N-terminal StrepII tag by Western blotting (B) prior to or after the digestion with Ulp1. Ribosome-bound NCs could be separated by centrifugation (RNC pellet) from the cleaved StrepII-Smt3-tag moiety (3xStrepII-Smt3) in the supernatant and the nascent chain was no longer detectable on the Western blot after Ulp1 cleavage. The faint signal at 52 kDa indicates a minor subpopulation of RNCs where the ester bond to the tRNA (NC-tRNA) was not hydrolyzed during sample preparation.
9.50 9.25 9.00 8.75 8.50 8.25 8.00 7.75 7.50 7.25 7.00 108
110
112
114
116
118
120
122
124
126
128 F1 (15N) [ppm]
F2 (1H) [ppm]
Fig. S2. Stable isotope-labeled background signals originating from ribosomes labeled during expression.½15N;1H-CRINEPT-HMQC experiment of a ribosome sample expressed and purified in absence of nascent chain substrate.
0 10 20 30 40 50 60 10
20 30 40 50 60
Residue number 1H linewidth [Hz]
20 30 40 50 60 70
Residue 1H linewidth [Hz]
E8β2
V10γ1
L11δ1 A12β
E18γ2 K19γ2
R22δ2 Q17γ2
R22δ3 V24β
K27β2
I31δ1
L34γ L34δ2
L35γ
V45γ2 V47γ1
V47γ2 R50δ2
R50δ3
A57β A56β V59γ2
B
Fig. S3. Line broadening analysis of SH3-RNCs indicative of nonspecific transient interactions between SH3 and the ribosome. Sequence-resolved1H line width analysis of cross peaks of (A)15N-1H-moieties and (B)13C-1H-moieties of both SH3-RNCs (Blue Circles) and SH3 cleaved from ribosomes (Red Circles). Filled circles indicate a considerable line width difference between SH3-RNCs and SH3 cleaved from ribosomes. Cross peaks, which are broadened beyond detection, are shown at the top above any value at the y-axis. Average1HNline width of SH3-RNCs (inBlue), SH3 cleaved from the ribosome (inRed), and SH3-F free in solution (inBlack) are plotted by dashed lines. In (B) the assignment of the individual13C-1H-moieties are given.
Fig. S4. NMR investigations of SH3-m10 RNCs.½13C;1H-HMQC spectra of (A) SH3-m10-F, (B) SH3-m10-RNCs, and (C) SH3-m10 cleaved from the ribosome. The peaks are labeled with single amino acid code in black. Background signals emanating presumably from the ribosome are shown as red R. Sequence-resolved
1H line width analysis of cross peaks of (D)15N-1H-moieties and (E)13C-1H-moieties of both SH3-m10-RNCs (Blue Circles) and SH3-m10 cleaved from the ribo-some (red circles). Filled circles indicate a considerable line width difference between SH3-m10-RNC and SH3-m10 cleaved from the riboribo-some. Cross peaks, which are broadened beyond detection, are shown at the top above any value at the y-axis. In (E) the assignment of the individual13C-1H-moieties are given. (F)
13Cαchemical shift deviation from the corresponding random coil chemical shift of SH3-m10-F free in solution versus the amino acid sequence. The lack of profound stretches of either up-fielded or down-fielded values is indicative of a random coil-like state of SH3-m10-F.
between SH3-T1-RNCs and SH3-T1-F added to the ribosomes. Cross peaks, which are broadened beyond detection are shown at the top above any value at the y-axis. In (H) the assignment of the individual13C-1H-moieties are given. (I) and (J) show sequence-resolved1H line width analysis of cross peaks of (I)
15N-1H-moieties and (J)13C-1H-moieties of both SH3-T2-RNCs (Blue Circles) and SH3-T2-F added to the ribosome (Red Circles). Filled circles indicate a consider-able line width difference between SH3-T2-RNCs and SH3-T2-F added to the ribosome sample. Cross peaks, which are broadened beyond detection, are shown
13 1 13 α
Fig. S6. Overview of the line broadening analysis.15N-1H moieties and13C-1H moieties that show line broadening in all four RNCs studied here (i.e. SH3, SH3-m10, SH3-T1, and SH3-T2) are highlighted in red, while moieties without a line broadening effect are highlighted in yellow. For the13C-1H moieties the exact side-chain moieties are listed.
Table S1. Translational diffusion of ribosome, SH3, and SH3-m10 nascent chains as well as SH3 and SH3-m10 cleaved from the ribosome
Sample
Diffusion of15N-filtered signal (SH3 or SH3-m10) (×10−7cm2∕s)
Diffusion of14N-filtered signal (ribosome) (×10−7cm2∕s)
SH3-RNCs 2.0 ± 0.4 1.4 ± 0.3
SH3-m10-RNCs 1.7 ± 0.4 1.5 ± 0.2
SH3 cleaved from ribosome 3.1 ± 0.2 n.d.
SH3-m10 cleaved from ribosome 3.3 ± 0.9 n.d.