Titration of the interaction between HEWL and hsp60. Spectra were recorded using a different sample for each titration point with a fixed concentration of 31 μM U-[2H, 15N], Val/Leu-[13CH3]proS, Ile-[13CH3]δ1 HEWL and a variation of the concentration of U-[2H, 15N], Met-[13CH3] hsp60. Each sample (40 μl) was loaded in a 1.7-mm tube, and the NMR experiments were recorded on a Bruker Avance III HD spectrometer operating at a 1H frequency of 850 MHz and equipped with a 1.7-mm TCI MicroCryoProbe. The 2D SOFAST-methyl-TROSY NMR (18) experiments were recorded at 75°C.

Thermal unfolding of HEWL with and without hsp60. The reversible thermal (un)folding of HEWL (41) was probed using two samples. HEWL reference sample: 31 μM HEWL U-[2H, 15N], Val/Leu-[13CH3]proS, Ile-[13CH3]δ1. HEWL with hsp60: 15 μM hsp60 (240 μM monomer concentration) U-[2H, 15N], Met-[13CH3] sample and 31 μM HEWL U-[2H, 15N], Val/Leu-[13CH3]proS, Ile-[13CH3]δ1 sample (two equivalents of HEWL per hsp60 particle). Each sample (40 μl) was loaded in a 1.7-mm tube, and the NMR experiments were recorded on a Bruker Avance III HD spectrometer operating at a 1H frequency of 850 MHz and equipped with a 1.7-mm TCI MicroCryoProbe. The 2D SOFAST-methyl-TROSY NMR experiments (18) were recorded from a temperature of 50° to 75°C with steps of 5°C between 50° and 60°C and steps of 2.5°C between 60° and 75°C. Percentage of folded state was extracted from the average ratio between the intensity of the signals in the folded and unfolded states for different residues.

Measurement of the translational diffusion coefficient of the free and hsp60-bound HEWL and MSG. For HEWL, three samples were used to acquire the 2D (13C,1H)-DOSY data sets (42) at a temperature of 65°C. Reference sample: 31 μM HEWL U-[2H, 15N], Val/Leu-[13CH3]proS, Ile-[13CH3]δ1; hsp60 reference sample: 15.5 μM hsp60 (250 μM monomer concentration) U-[2H, 15N], Met-[13CH3]; and hsp60 with HEWL: 15,5 μM hsp60 (250 μM monomer concentration) U-[2H, 15N], Met-[13CH3] and 31 μM HEWL U-[2H, 15N], Val/Leu-[13CH3]proS, Ile-[13CH3] δ1 sample (two equivalents of HEWL per hsp60 particle).

For MSG, three samples were used to acquire the 2D (13C,1H)-DOSY data sets. As thermally unfolded MSG is not soluble, the MSG reference spectrum was acquired at 50°C with 31 μM folded MSG U-[2H, 15N], Ile-[13CH3] δ1. For the sample containing unfolded MSG bound to hsp60, 15.5 μM hsp60 (250 μM monomer concentration) U-[2H, 15N], Met-[13CH3] was mixed to 31 μM MSG U-[2H, 15N], Ile-[13CH3] δ1. MSG was thermally unfolded at 60°C in the presence of hsp60 (two equivalents of MSG per hsp60 particle), and DOSY (Diffusion Ordered SpectroscopY) spectra were acquired at 60°C. The hsp60 reference sample spectrum was acquired at 60°C with a sample containing 15.5 μM hsp60 (250 μM monomer concentration) U-[2H, 15N], Met-[13CH3].

Paramagnetic labeling of HEWL and MSG for the detection of intermolecular paramagnetic relaxation enhancement (PRE) effects. Spin labeling of the ε-amino groups of the solvent accessible HEWL (Sigma-Aldrich) or MSG lysine residues with OXYL-1-NHS (1-oxyl-2,2,5,5-tetramethylpyrolline-3-carboxylate-N-hydroxysuccimide ester, Toronto Research Chemicals) was carried out following a published protocol (43). Protein was dissolved in the labeling buffer [10 mM Na2CO3 (pH 9.2)] at a concentration of 40 μM. A stock solution was prepared by dissolving 10 mg of OXYL-1-NHS in 100 μl of dimethyl sulfoxide. After addition of a sixfold molar excess of OXYL-1-NHS over lysine residues to the protein solution, the reaction mixture was incubated for 1 hour at room temperature, followed by 4 hours at 4°C. The excess of spin label was removed by washing the sample with approximately 20 volumes of NMR buffer using centrifugal filter devices (Vivaspin 15, 5000 MWCO, Vivascience). Homogeneity of the lysine labeling was checked by mass spectrometry.

We measured paramagnetic relaxation (PRE) enhancement values, to provide qualitative information on the interactions between HEWL or MSG and hsp60, by comparing peak intensities in 2D SOFAST-methyl-TROSY NMR experiments recorded at 75°C with a 16 μM hsp60 (256 μM monomer concentration) U-[2H] Met-[13CH3], Val-[13CH3]proS in interaction with either 32 μM OXYL-1-NHS–labeled 1H HEWL (Iox)/MSG (Iox) or reference diamagnetic HEWL (Ired)/MSG (Ired). The reference sample was obtained by reducing the spin label of the OXYL-1-NHS–labeled sample by incubation with sodium ascorbate (2.2 mM) for 24 hours at 4°C. NMR data were recorded using a Bruker Avance III HD spectrometer operating at a 1H frequency of 950 MHz and equipped with a 5-mm cryogenically cooled pulsed-field gradient triple-resonance probes.

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