Abstract
Despite the biological and pharmaceutical significance of membrane proteins, their tertiary structures constitute less than 3% of known structures. One of the major obstacles for initiating structural studies of membrane proteins by NMR spectroscopy is the generation of high amounts of isotope-labeled protein. In this work we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length, and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement, and has the potential to facilitate an increase in the number of structures of single-pass TMDs, which are difficult to solve by other means.
PMID: 26309151 [PubMed - as supplied by publisher]
[NMR paper] Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.
Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.
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Chemistry. 2015 Jul 28;
Authors: Didenko T, Proudfoot A, Dutta SK, Serrano P, Wüthrich K
Abstract
High-resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments...
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[NMR paper] Efficient long-distance NMR-PRE and EPR-DEER restraints for two-domain protein structure determination.
Efficient long-distance NMR-PRE and EPR-DEER restraints for two-domain protein structure determination.
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Protein Cell. 2013 Nov 27;
Authors: Wu K, Shi C, Li J, Wang H, Shi P, Chen L, Wu F, Xiong Y, Tian C
PMID: 24282082
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[NMR paper] Structural Elucidation of Transmembrane Transporter Protein Bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy.
Structural Elucidation of Transmembrane Transporter Protein Bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy.
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Biochim Biophys Acta. 2013 Jun 14;
Authors: Choudhury AR, Perdih A, Zuperl S, Sikorska E, Solmajer T, Jurga S, Zhukov I, Novi? M
Abstract
Membrane proteins...
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Structural Elucidation of Transmembrane Transporter Protein Bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy
Structural Elucidation of Transmembrane Transporter Protein Bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy
Publication date: Available online 14 June 2013
Source:Biochimica et Biophysica Acta (BBA) - Biomembranes</br>
Author(s): Amrita Roy Choudhury , Andrej Perdih , Špela Župerl , Emilia Sikorska , Tom Solmajer , Stefan Jurga , Igor Zhukov , Marjana Novi?</br>
Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing...
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06-15-2013 08:18 AM
[NMR paper] NMR structure determination of a membrane protein with two transmembrane helices in m
NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system.
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Biochemistry. 2005 Apr 5;44(13):5196-206
Authors: Howell SC, Mesleh MF, Opella SJ
The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that...
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11-25-2010 08:21 PM
[NMR paper] Production in two-liter beverage bottles of proteins for NMR structure determination
Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N.
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J Struct Funct Genomics. 2004;5(1-2):87-93
Authors: Zhao Q, Frederick R, Seder K, Thao S, Sreenath H, Peterson F, Volkman BF, Markley JL, Fox BG
The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology...
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Efficient protein production method for NMR using soluble protein tags with cold shoc
Efficient protein production method for NMR using soluble protein tags with cold shock expression vector
Abstract The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins...
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Efficient protein production method for NMR using soluble protein tags with cold shoc
Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.
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J Biomol NMR. 2010 Sep 16;
Authors: Hayashi K, Kojima C
The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To...
Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy.
Linear Mode
