Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.
 

Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.

Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.

Bio Protoc. 2015 Jul 20;5(14)

Authors: Horst R, Wüthrich K

Abstract
Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck that slows down the structural biology of IMPs is the preparation of diffracting crystals or concentrated solutions of stable isotope labeled IMPs. Here, we describe procedures to monitor the quality of aqueous solutions of [(2)H, (15)N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of ?-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with ?-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al., 2013). 2D [(15)N, (1)H]-correlation maps are used as "fingerprints" to assess the foldedness of the IMP in solution. For promising samples, these "inexpensive" data are then supplemented with measurements of the translational and rotational diffusion coefficients, which give information on the shape and size of the IMP/detergent mixed micelles. Using microcoil equipment for these NMR experiments enables data collection with only micrograms of protein and detergent. This makes serial screens of variable solution conditions viable, enabling the optimization of parameters such as the detergent concentration, sample temperature, pH and the composition of the buffer.


PMID: 27077076 [PubMed - as supplied by publisher]



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