Optimization of protein NMR sample buffer conditions

After optimize the protein expression conditions, and get the good yield of the protein, for the next NMR studies, one still needs to optimize  the protein NMR buffer conditions, in order to avoid protein aggregation, (which can be caused by the inter-protein electrostatic interaction or the presence of hydrophobic patches on the protein surface,) slow precipitation, degradation, and others, e.g. , mixture of folding and unfolding, multiple populations, etc. So the next suggestions can be considered:

1. Look into literature first (e.g. x-ray structure papers) and find out a similar one (especially, pH and salt concentration) as your starting NMR buffer condition, if there is no publication for your new protein yet, try to search other proteins’, which are in the same family of yours.

2. Before start a NMR exp., you’d better use analytical ultracentrifugation (Sedimentation) method or dynamic light scattering (DLS) to screen pH (e.g. from 4.5, 5, 5.5, to 7.5) and salt concentration (e.g. from 0, 50, 100, 150, to 450mM), and experimentally prove that your protein is uniformly under the monomer (dimer, trimer, or tetramer, etc.) mono-disperse   state in your chosen NMR buffer condition ( rather  than a multi-disperse mixture).

3. For homonuclear NMR exp., Na/K phosphate (pH5.0 to 7.5), Tris-d11 (pH7.5 to 8.5), or non-proton containing compounds can be used as the buffer; for heteronuclear NMR exp., protonated buffers are fine.

4. In general, around physiological pH (6-7) and salt (100-150 mM) conditions are the best, low pH (< 6) can give good quality spectra due to slow NH exchange, but the protein structures could be influenced too.

5. ~5mM DTT, ~5mM EDTA, and ~0.02% NaN3 are commonly included in the buffer.  in addition, ~10mM CHAPS, a non-denaturing detergent, may prevent the protein aggregation caused by hydrophobic interaction (J. Biomol. NMR, 1993, 3, 121-126.), and Glycine (Molar scaled) may stabilize the protein and prevent from heat-denaturing.

6. For both 600 and 800 MHz spectrometers equipped with cold probes, at least 0.05mM of your N15 labeled protein sample is needed for 2D HSQC, and at least 0.35mM of your C13/N15 labeled protein sample is needed for other backbone assignments 3D triple-resonance exps.


updated on Sept. 18, 2014

edited on Jan. 20, 2014

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