3D DOSY-TROSY to determine the translational diffusion coefficient of large protein complexes.
The translational diffusion coefficient is a sensitive parameter to probe conformational changes in proteins and protein-protein interactions. Pulsed-field gradient NMR spectroscopy allows one to measure the translational diffusion with high accuracy. Two-dimensional (2D) heteronuclear NMR spectroscopy combined with diffusion-ordered spectroscopy (DOSY) provides improved resolution and therefore selectivity when compared with a conventional 1D readout. Here, we show that a combination of selective isotope labelling, 2D ¹H-¹³C methyl-TROSY (transverse relaxation-optimised spectroscopy) and DOSY allows one to study diffusion properties of large protein complexes. We propose that a 3D DOSY-heteronuclear multiple quantum coherence (HMQC) pulse sequence, that uses the TROSY effect of the HMQC sequence for ¹³C methyl-labelled proteins, is highly suitable for measuring the diffusion coefficient of large proteins. We used the 20 kDa co-chaperone p23 as model system to test this 3D DOSY-TROSY technique under various conditions. We determined the diffusion coefficient of p23 in viscous solutions, mimicking large complexes of up to 200 kDa. We found the experimental data to be in excellent agreement with theoretical predictions. To demonstrate the use for complex formation, we applied this technique to record the formation of a complex of p23 with the molecular chaperone Hsp90, which is around 200 kDa. We anticipate that 3D DOSY-TROSY will be a useful tool to study conformational changes in large protein complexes.
Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Tatiana
T
Didenko
Rolf
R
Boelens
Stefan G D
SG
Rüdiger
D004058:Diffusion; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D000071876:Prostaglandin-E Synthases; D011487:Protein Conformation; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods
D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D010750:Phosphoproteins; C490849:PTGES3 protein, human; D000071876:Prostaglandin-E Synthases