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]
Paramagnet Induced Signal Quenching in MAS-DNP Experiments in Homogeneous Solutions
From The DNP-NMR Blog:
Paramagnet Induced Signal Quenching in MAS-DNP Experiments in Homogeneous Solutions
Corzilius, B., et al., Paramagnet Induced Signal Quenching in MAS-DNP Experiments in Homogeneous Solutions. J. Magn. Reson., (0).
http://www.sciencedirect.com/science/article/pii/S1090780713003042
nmrlearner
News from NMR blogs
0
02-05-2014 06:08 PM
[NMR paper] The micro-scale synthesis of (117)Sn-enriched tributyltin chloride and its characterization by GC-ICP-MS and NMR techniques.
The micro-scale synthesis of (117)Sn-enriched tributyltin chloride and its characterization by GC-ICP-MS and NMR techniques.
Related Articles The micro-scale synthesis of (117)Sn-enriched tributyltin chloride and its characterization by GC-ICP-MS and NMR techniques.
Chemosphere. 2014 Jan 25;
Authors: Peeters K, Iskra J, Zuliani T, S?an?ar J, Mila?i? R
Abstract
Organotin compounds (OTCs) are among the most toxic substances ever introduced to the environment by man. They are common pollutants in marine ecosystems, but are also present in the...
nmrlearner
Journal club
0
01-30-2014 05:38 PM
[NMR paper] Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy.
Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy.
Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy.
J Biomol NMR. 2013 Aug 21;
Authors: Mote KR, Gopinath T, Veglia G
Abstract
The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy...
nmrlearner
Journal club
0
08-23-2013 01:07 AM
Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems
From The DNP-NMR Blog:
Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems
Cheng, C.Y. and S. Han, Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems. Annu Rev Phys Chem, 2013. 64(1): p. 507-32.
http://www.ncbi.nlm.nih.gov/pubmed/23331309
nmrlearner
News from NMR blogs
0
07-08-2013 02:17 PM
Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles
Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles
Abstract Optimization of aqueous solutions of the integral membrane protein (IMP) OmpW for NMR structure determination has been monitored with micro-coil NMR, which enables the acquisition of NMR spectra using only micrograms of protein and detergent. The detergent 30-Fos (2-undecylphosphocholine) was found to yield the best 2D -TROSY correlation NMR spectra of -labeled OmpW. For the OmpW structure determination we then optimized the 30-Fos concentration,...
nmrlearner
Journal club
0
08-16-2012 09:18 AM
Monitoring H-D exchange on second time scale with Frydman NMR spectroscopy
http://www.bionmr.com/images/frydman.gif
Real-Time Monitoring of Chemical Transformations by Ultrafast 2D NMR Spectroscopy
Maayan Gal, Mor Mishkovsky, and Lucio Frydman
J. Am. Chem. Soc.; 2006; 128(3) pp 951 - 956;
Abstract:
Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.
Linear Mode
