1 / 11

Protein Sample Requirements for HTP NMR Spectroscopy

Protein Sample Requirements for HTP NMR Spectroscopy. NIGMS Protein Structure Initiative Protein Production Workshop (March 2002). Demand Profile: NMR samples. Concentration: 0.5 – 2 mM 13 C/ 15 N-double labeled Monodisperse (no aggregation) Highly purified (> 95%)

adara
Download Presentation

Protein Sample Requirements for HTP NMR Spectroscopy

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related