Related ArticlesIsotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Methods Enzymol. 2015;565:193-212
Authors: Fan Y, Emami S, Munro R, Ladizhansky V, Brown LS
Abstract
Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly. We have demonstrated that, by using isotopic labeling in yeast Pichia pastoris, one can inexpensively produce milligram quantities of doubly labeled functional samples, which yield multidimensional ssNMR spectra of high resolution suitable for detailed structural investigation. This was achieved by combining protocols of economical isotope labeling of soluble proteins previously used for solution NMR with protocols of expression of eukaryotic membrane proteins successfully employed for other methods. We review two cases of such isotope labeling, of fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1.
PMID: 26577733 [PubMed - as supplied by publisher]
An economic approach to efficient isotope labeling in insect cells using homemade 15 N-, 13 C- and 2 H-labeled yeast extracts
An economic approach to efficient isotope labeling in insect cells using homemade 15 N-, 13 C- and 2 H-labeled yeast extracts
Abstract
Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein 15N and 13C labeling of up...
nmrlearner
Journal club
0
06-12-2015 07:48 PM
[NMR paper] Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.
Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.
Related Articles Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.
J Biomol NMR. 2015 May 9;
Authors: Baker LA, Daniëls M, van der Cruijsen EA, Folkers GE, Baldus M
Abstract
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...
nmrlearner
Journal club
0
05-10-2015 07:49 PM
Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling
Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling
Abstract
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...
nmrlearner
Journal club
0
05-08-2015 09:18 PM
[NMR paper] Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
Related Articles Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
Chembiochem. 2014 Nov 12;
Authors: Schubeis T, Lührs T, Ritter C
Abstract
We present an efficient method for the reduction of spectral complexity in the solid-state NMR spectra of insoluble protein assemblies, without loss of signal intensity....
nmrlearner
Journal club
0
11-14-2014 08:40 PM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
nmrlearner
Proteins
0
01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
nmrlearner
Journal club
0
01-21-2011 01:22 AM
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...
nmrlearner
Journal club
0
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...
Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Linear Mode
