RESOURCES

Name of Method: NMR

Result expected: 5 lines. Summarise the results that can be expected.
NMR spectroscopy for protein-protein complex characterization can be exploited for:

• screening of complex formation
• Characterisation of the complex:
• The first point can be rapidly assessed through the ¹H- ¹⁵N HSQC spectra and the analysis provides information on the kinetic and thermodynamic properties of the complex in solution (complex lifetime and equilibrium dissocation constant). This analysis is required before proceeding to the second point. A rough information whether the complex is formed can also be obtained on unlabelled samples in case one protein is small (<10kDa) and the other is large (>50kDa).
• The second point structurally characterizes the complex and can be addressed by chemical shift mapping, intermolecular filtered NOEs and RDC, which can be differently used depending on the information obtained at point 1. Heteronuclear relaxation rates provide precise information on the aggregation state.

K_D range:
NMR can characterize interactions with K_D < 10-3M which is an extremely low limit.

MW range: The screening can be performed for high MW complexes in principle up to 900kDa, while the limit for structure characterization is about 50kDa.

Purity: What level is needed: At least 90%

Accuracy: (Da) NMR has atomic resolution

Quantity required: For a complex AB, assuming that A is the 'receptor' and B the 'ligand' how much protein is needed to characterize the complex fully (e.g. obtain K_D or get MW) ?
3-5mg per screen (can screen 10 at a time). One of the partners should be labeled.

HTP: Is the method HTP (can it use 96-well sample plates in an automated fashion)? Screening of a complex is a medium throughput screening system, while the structural characterization of complexes is not HTP.

Speed: Will the user have a quantitative answer immediately after collecting the data?
Yes for point 1 (KD determination a titration and some analysis is needed) and no for point 2.

Cost: (approximate cost of consumables per ONE experiment) Only the cost of the protein

Access: Is an instrument available (name all SPINE2 sites you know)? Florence, Utrecht, Stockholm

User expertise: Can the method be provided as a service (measurement and data analysis done by local staff) or only access and assistance on a collaborative basis?
Florence and Utrecht are equipped for both types of access.

Special requirements: Any special sample needs?
15N labelling samples are needed for screening
13C, 15N labeling samples are needed for characterisation of complexes, also 2H labeling for systems with MW>30kDa.