Cambridge Isotope Laboratories, Inc. (CIL) is pleased to offer new and exciting isotope-enriched proteins for use as standards in NMR spectroscopy. Isotope-enriched protein standards are ideal for:
• Aiding in the development of new pulse sequences
• Optimizing parameters for a given pulse sequence
• Assessing spectrometer performance
• Training purposes
High-quality, high-purity standards from Nexomics are invaluable tools for bioNMR. Each product is accompanied by the following data:
• 1 H-15N HSQC (15N-labeled proteins)
• 1 H-13C HSQC (13C-labeled proteins)
• CO-NH projection of 3D HNCO ( 15N,13C-labeled proteins)
• SDS PAGE (for all labeled proteins)
• MALDI-TOF (for all labeled proteins)
• 15N-edited X-filtered 2D NOESY (NEX-XF1)
Maltose Binding Protein (NEX-MBP)
NEX-MBP is a 44.9 kDa monomeric protein for which multiple sets of resonance assignments (BMRB database) and 3D structures (PDB database) are publicly available. This product is uniformly 2H,15N,13C-enriched with selective incorporation of protons into methyl groups of Ile-δ1, Leu-δ and Val-γ side chains. As nonuniform sampling (NUS) and other NMR techniques emerge to push the size limitations of NMR to new boundaries, large protein standards, such as NEX-MBP, will be required to test data-collection and processing strategies.
NEX-MBP sample formulations:
NEX-MBP1: Apo Conformation 0.5 mM 2H,15N,13C and ILV methyl 1H,13C MBP in 10% D2O, 0.02% NaN3, 20 mM sodium phosphate @ pH 7.2
NEX-MBP2: Closed Conformation 0.5 mM 2H,15N,13C and ILV methyl 1H,13C MBP with 3 mM maltotriose, 10% D2O, 0.02% NaN3, 20 mM sodium phosphate @ pH 7.2
NEX-MBP3: Open Conformation 0.5 mM 2H,15N,13C and ILV methyl 1H,13C MBP with 2 mM β-cyclodextrin, 10% D2O, 0.02% NaN3, 20 mM sodium phosphate @ pH 7.2
IMAGE: “OPEN” (BLUE) AND “CLOSED” (RED) SUPERPOSITION
In an X-filtered experiment, only NOEs between 15N/13C-1H and 14N/12C-1H (e.g. interchain NOEs) protons are observed. NOEs between protons connected to 15N,13C are filtered (intrachain NOEs). When uniformly double-labeled protein sample is mixed with a naturalabundance protein sample, the interface will give rise to the only observable NOESY cross peaks. This powerful strategy enables the spectroscopist to discern
intra from inter NOESY cross peaks, thereby providing essential distance constraints for defining the dimer interface (Lee, et al., 1994, 350:87; Palmer, et al., 1991, 93:151; Schleucher, et al., 1994, 4:301).
NEX-XF1 is a 16 kDa protein (A. fulgidus antitoxin vapB21 homodimer) for which a set of resonance assignments (bmr7362), 3D structure (2NWT) and other NMR data are available in the public domain. This is a mixture of unlabeledand uniformly 15N,13C-enriched protein (25% homodimer unlabeled; 50% heterodimer unlabeled/labeled; 25% homodimer labeled) and is perfect to set up X-filtered NOESY experiments.
NEX-XF1 homodimer sample formulation:
NEX-XF1: 13C,15N-labeled and unlabeled sample conditions1 mM protein, 20 mM NH4OAc pH 5.5, 100 mM NaCl, 5 mM CaCl2, 10% D2O, 0.02 % NaN3
NEX-UB1 is a small 8.8 kDa monomeric protein for which multiple sets of resonance assignments (BMRB database) and 3D structures (PDB database) are publicly available. This protein standard is uniformly 15N, 13C-enriched. Ubiquitin has been used as an industry-wide standard in the protein NMR field for many years. NEX-
UB1 sample formulation: NEX-UB1: Uniformly 15N, 13C-labeled ubiquitin in 90% H2 O; 10% D2 O 10 mM sodium phosphate buffer, pH 6.5
e development of new pulse sequences
experiments.