Protein Sample Requirements for HTP NMR Spectroscopy

1. Protein Sample Requirements for HTP NMR Spectroscopy NIGMS Protein Structure Initiative Protein Production Workshop (March 2002)

2. Demand Profile: NMR samples • Concentration: 0.5 – 2 mM • 13C/15N-double labeled • Monodisperse (no aggregation) • Highly purified (> 95%) • No paramagnetic impurities (NMR Line Broadening) • No solid particles (B-field homogeneity) • Long-term stability (up to several weeks)  All at pH < 7.5 and about 25 C

3. Stable Isotope Labeling Mandatory • Sample Screening:15N-labeling (~ $30 / L Medium) • Assignment and Structure Determination:15N/13C-labeling (~$500 / L Medium) for molecular weights < ~ 25 kDa 15N/13C/2H-labeling (~$1000 / L Medium) for molecular weights > ~ 25 kDa • Media: Minimal with glucose as sole carbon source or labeled full media • Cell-free systems

4. 2D N,H HSQC: One Signal per Amino Acid Residue From: Montelione et al. (2000) Nature Struc. Biol. 7 (Suppl.) 982.

5. Amount of Protein Required • Sample volume: ~500 mL • Molecular weight: 8-30 kDa •  between 2 and 30 mg of protein (typically around 10 mg) • Use of “Shigemi” tubes (350 mL) can reduce the required amount by about 40%

6. Long-term Stability of NMR samples • Choose conditions preventing (slow) precipitation • Removal of proteases:PurificationChoice of Expression SystemAdd protease inhibitor “cocktail” • Suppression of bacterial growth: Add 10-50 mM NaN3 Micro-filtration as final purification step (D2O as solvent) • Prevent oxidation of solvent exposed SH groups: addition of (deuterated) DTT

7. Adjustment of pH • Main constraint: amide proton exchange with bulk water protons is too fast above pH ~ 7.5 • pH ~ 6.5 is a good compromise: NH exchange conveniently reduced, but side chains mostly in physiological state of protonation • Buffer: no 1H-containing components • pI of protein needs to be considered to maintain high solubility

8. Adjustment of Ionic Strength • Ionic Strength < 500 mM when working with conventional probes [Adjustment of r.f. circuit of probe – “tuning and matching”] • Ionic Strength < 100 mM (preferably ~ 50 mM) when working with cryogenic probes [High Q-factors of cryo r.f. coils are rapidly degraded at high salt concentration] • Key parameter for high solubilty

9. Tags for Affinity Chromatography • Target protein needs to be cleaved from fusion constructs (such as MBP): size limitation of NMR • Cleavage of short tags (His,Strep…) is advantageous because no (additional) intense signals of flexibly disordered polypeptide segments are introduced. • Tags removed by proteases may lead to the challenge to quantitatively remove proteases. • Hetero-nuclear multidimensional NMR can “live” with short tags, though they may require attention during data processing.

10. Biophysical Characterization of NMR samples • Isotope Labeling: Mass Spectrometry • Mono-disperse solution: Dynamic Light Scattering (Ultra-centrifugation) • Foldedness: CD / 2D N,H-HSQC NMR • Long-term stability: 2D N,H-HSQC NMR

11. Robotic Systems • None available