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 [2H, 15N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of β-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with α-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al., 2013). 2D [15N, 1H]-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.
Keywords: Micro-scale NMR, Structural Biology, Integral Membrane Proteins, Transverse relaxation optimized spectroscopy (TROSY), NMR sample optimization
Materials and Reagents
Studies of IMPs
Equipment
NMR data collection
Procedure
Data analysis
Processing and analysis of NMR datasets: Process all NMR experiments with the Bruker standard software Topspin. For all experiments, the data matrices are multiplied with an exponential window function in the 1H-dimension, and for 2D [15N, 1H]-TROSY a 75°-shifted sine bell window (De Marco and Wüthrich, 1976) is applied in the 15N-dimension The 2D [15N, 1H]-TROSY data sets were analyzed using the XEASY module (Bartels et al., 1995) of the CARA release 1.5.5 (www.cara.nmr.ch). The 1H-TRO-STE datasets are analyzed using the Bruker T1/T2-software package, as described in the Topspin DOSY application manual, chapter 3.2. The TROSY and anti-TROSY components of the TRACT data set are first separated using the Bruker standard AU program split, and then individually integrated using the Bruker integration module. The integrals were fitted to a mono-exponential decay, using the program XMGRACE (http://plasma-gate.weizmann.ac.il).
Notes
Optimization of NMR acquisition parameters for the 2D [15N, 1H]-TROSY, 1H-TRO-STE and TRACT experiments:
Recipes
Acknowledgments
This work was adapted from previously published studies on the E. coli outer membrane protein X (OmpX) (Stanczak et al., 2009), and was used as a platform for the structure determination of E. coli OmpW (Horst et al., 2014). The procedures described in this protocol were also used to characterize E. coli OmpA in lipid bilayer nanodiscs and detergent micelles (Susac et al., 2014). This work was supported by the Roadmap initiative grant P50 GM073197 for technology development. Kurt Wüthrich is the Cecil H. and Ida M. Green Professor of Structural Biology at the Scripps Research Institute.
References
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