Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.
Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization
From The DNP-NMR Blog:
Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization
Yamamoto, K., et al., Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015. 1848(1, Part B): p. 342-349.
http://dx.doi.org/10.1016/j.bbamem.2014.07.008
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[NMR paper] Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization.
Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization.
Related Articles Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization.
Biochim Biophys Acta. 2014 Jul 10;
Authors: Yamamoto K, Caporini MA, Im SC, Waskell L, Ramamoorthy A
Abstract
While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is...
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07-16-2014 10:46 AM
Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization
Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization
Publication date: Available online 11 July 2014
Source:Biochimica et Biophysica Acta (BBA) - Biomembranes</br>
Author(s): Kazutoshi Yamamoto , Marc A. Caporini , Sang-Choul Im , Lucy Waskell , Ayyalusamy Ramamoorthy</br>
While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR...
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
Optimization of amino acid type-specific (13)C and (15)N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.
J Biomol NMR. 2010 Dec 18;
Authors: Hefke F, Bagaria A, Reckel S, Ullrich SJ, Dötsch V, Glaubitz C, Güntert P
We present a computational method for finding optimal labeling patterns for the backbone...
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12-21-2010 01:00 PM
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
Abstract We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273â??6279 (1982)), types of amino acids are labeled with 13C or/and 15N such that cross peaks between 13CO(i â?? 1) and 15NH(i) result only for pairs...
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12-21-2010 02:14 AM
[NMR paper] How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
Related Articles How to prepare membrane proteins for solid-state NMR: A case study on the alpha-helical integral membrane protein diacylglycerol kinase from E. coli.
Chembiochem. 2005 Sep;6(9):1693-700
Authors: Lorch M, Faham S, Kaiser C, Weber I, Mason AJ, Bowie JU, Glaubitz C
Several studies have demonstrated that it is viable to use microcrystalline preparations of water-soluble proteins as...
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12-01-2010 06:56 PM
[NMR paper] Selective and extensive 13C labeling of a membrane protein for solid-state NMR invest
Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations.
Related Articles Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations.
J Biomol NMR. 1999 May;14(1):71-4
Authors: Hong M, Jakes K
The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the...
Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling
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