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 (gt 95)
• No paramagnetic impurities (NMR Line Broadening)
• No solid particles (B-field homogeneity)
• Long-term stability (up to several weeks)
• ? All at pH lt 7.5 and about 25 C

3
Stable Isotope Labeling Mandatory

• Sample Screening 15N-labeling ( 30 / L
  Medium)
• Assignment and Structure Determination 15N/13C-l
  abeling (500 / L Medium) for molecular weights
  lt 25 kDa
• 15N/13C/2H-labeling (1000 / L Medium) for
  molecular weights gt 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 Purification Choice of
  Expression System Add 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 lt 500 mM when working with
  conventional probes Adjustment of r.f. circuit
  of probe tuning and matching
• Ionic Strength lt 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