Nuclear magnetic resonance (NMR) spectroscopy

NMR spectra were recorded at 298 K on a Bruker 500 MHz (Boston University School of Medicine) or on a Bruker 800 MHz (Brandeis University) magnet, each equipped with a cryoprobe. Backbone resonance assignments of eIF2α-NTD, eIF2α-NTD_S51D, and eIF2α-CTD were obtained using standard triple-resonance experiments on ²H/¹⁵N/¹³C-labeled samples at 300 mM NaCl.

Samples for NMR measurements were prepared in Buffer A150 (10 mM Na-phosphate (pH 7), 150 mM NaCl, 0.01% NaN₃, 2 mM DTT, 1 mM EDTA, 0.1 mM AEBSF) with 5% ²H₂O. Transverse relaxation optimized ¹H–¹⁵N-heteronuclear single-quantum coherence (TROSY-HSQC) spectra of ²H/¹⁵N-labeled eIF2α, eIF2α_S51D, eIF2α-NTD and eIF2α-NTD_S51D were collected in the absence and presence of excess eIF2Bα or eIF2Bβ and compared. TROSY-HSQC spectra of ²H/¹⁵N/¹³C-labeled eIF2α-CTD were collected in the presence and absence of excess eIF2Bα or eIF2Bβ and compared. HSQC spectra of ²H/¹⁵N-labeled eIF2α_S51D both free and in the presence of eIF2Bα were also collected.

NMR chemical shift perturbation (CSP) assays were performed in combination with deletion analysis to map the intramolecular surface, and in combination with site-directed mutagenesis to determine the effects of phosphorylation, as previously described (27–30). When comparing spectra of the various constructs in the absence and presence of excess eIF2Bα or eIF2Bβ, a pattern of selective signal loss in peaks belonging to interface residues was observed upon binding. The selective signal loss is due to cross-relaxation between the free and bound state, a phenomenon observed in cases with a weak interaction in which the labeled protein is in fast equilibrium between a free state and a complex that is too large to observe by NMR (31). A statistical analysis of both chemical shift perturbation and signal loss was performed by calculating average change and standard deviation for all peaks on the protein, as described previously (27). Peak movement, or decrease in intensity, by more than two standard deviations from the average was considered statistically significant. Changes between one and two standard deviations were also mapped on the protein structures.