Buffer Optimization for NMR Samples Using Microprobe

Rossi et al. J. Biomolecular NMR Supplementary Revised Oct14, 2009

SUPPLEMENTARY MATERIAL

A Microscale Protein NMR Sample Screening Pipeline

Paolo Rossi1,*, G.V.T. Swapna1,*, Yuanpeng J. Huang1, James M. Aramini1,

Clemens Anklin2, Kenith Conover1,Keith Hamilton1, Rong Xiao1, Thomas B. Acton1, Asli Ertekin1, John K. Everett1, and Gaetano T. Montelione1,3,**

1Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, U.S.A and Northeast Structural Genomics Consortium.

2Bruker Biospin Corporation, 15 Fortune Drive, Billerica MA 01821, USA.

3Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway, NJ 08854, U.S.A.

*These authors contributed equally to this work and should be recognized as co-first authors.

Buffer optimization for NMR samples using microprobe

Buffer preparation: MJ001-MJ012
Purify protein with Ni-NTA affinity column, optionally followed by gel filtration chromatography.
Concentrate protein samples to ~ 1.0 - 1.5 mM in elution buffer
Change to buffer MJ001-MJ012 using desalting plate
Measure concentration using nanodrop
Transfer samples to microtube (1 or 1.7 mm)
Monitor the samples and recorder the status
Store the samples at 4 C for one week
Monitor the samples and record the status again
Direct the samples to NMR screening

Supplementary Table S1. NMR buffers used for buffer optimization screening.

Buffer ID,type / pH / Recipe
Ni-NTA* elution / 7.5 / 50 mM Tris**, 500 mM NaCl, 500 mM Imidazole, 1 mM TCEP¶, 0.02% NaN3, 5% D2O
MJ001 / 6.5 / 20 mM MES†, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT‡, 0.02% NaN3, protease inhibitor# 1x, 5% D2O
MJ002 / 5.5 / 20 mM NH4OAc, 100 mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ003 / 4.5 / 20mM NH4OAc, 100mM NaCl, 5mM CaCl2, 10mM DTT, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ004 / 5.0 / 50 mM NH4OAc, 10 mM DTT, 50 mM Arginine, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ005 / 5.0 / 50 mM NH4OAc, 10 mM DTT, 5% CH3CN, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ006 / 6.0 / 50 mM MES, 10 mM DTT, 50 mM Arginine, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ007 / 6.0 / 50 mM MES, 10 mM DTT, 5% CH3CN, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ008 / 6.5 / 25 mM Na2PO4, 450 mM NaCl, 10 mM DTT, 20 mM ZnSO4, protease inhibitor 1x, 0.01% NaN3 (pH 6.5), 5% D2O
MJ009 / 6.5 / 20 mM MES, 100 mM NaCl, 5% CH3CN, 10mM DTT, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ010 / 6.5 / 20 mM MES, 100 mM NaCl, 50 mM Arginine, 10mM DTT, 0.02% NaN3, protease inhibitor 1x, 5% D2O
MJ011 / 6.5 / 20 mM MES, 100 mM NaCl, 1% Zwitter§, 10 mM DTT, 0.02% NaN3, 5% D2O
MJ012 / 6.5 / 20 mM MES, 100 mM NaCl, 50 mM ZnSO4, 10 mM DTT, 0.02% NaN3, protease inhibitor 1x, 5% D2O

*Ni-NTA: Superflow Ni-charged resin (QIAGEN), ‡DTT: Dithiothreitol, †MES: 2-(N-morpholino)ethanesulfonic acid, §Zwitter: ZWITTERAGENT® 3-12 Detergent cat. 963015 (CALBIOCHEM), ¶TCEP: tris(2-carboxyethyl)phosphine, **Tris: tris(hydroxymethyl)aminomethane, #Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE).

Supplementary Table S2. Table of rotation correlation times for sixteen monomeric proteins, determined by 15N relaxation measurement.

Protein name /
NESG ID / Number of residues / MW (kDa) / c (ns)
B-domain / 60 / 7.2 / 4.05
PsR76A / 61 / 7.2 / 5.1
Ubiquitin / 76 / 9.0 / 4.4
TR80 / 86 / 10.5 / 7.0
HR2873B / 88 / 10.7 / 5.7
DvR115G / 94 / 10.9 / 6.5
VfR117 / 87 / 11.2 / 6.3
MrR110B / 104 / 11.8 / 7.8
VpR247 / 108 / 12.5 / 8.1
BcR97A / 105 / 13.1 / 8.8
SyR11 / 115 / 12.4 / 7.1
PfR93A / 114 / 13.6 / 9.0
SoR190 / 118 / 13.8 / 7.7
ER541-37-162 / 135 / 15.8 / 10.0
ER540 / 168 / 18.8 / 11.3
MvR76 / 166 / 20.2 / 12.2

Fig. S1. Labor intensive steps have been moved to robotic handlers: A) Gilson 96-well filling station transfer samples from Eppendorf tubes to microtubes; B) Bruker B-ACS 60, a sixty position sample changer for automated run using Bruker IconNMR; C) 1-mm Bruker TXI 1 Microprobe; D) 1.7-mm Bruker TCI 1.7 MicroCryoprobe used for data collection on a Bruker Avance 600 MHz spectrometer.