Repository logo
 

Integral membrane protein structure determination using pseudocontact shifts.


Change log

Authors

Crick, Duncan J 
Wang, Jue X 
Graham, Bim 
Swarbrick, James D 
Mott, Helen R 

Abstract

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

Description

Keywords

Archaeal Proteins, Halorhodopsins, Lanthanoid Series Elements, Membrane Proteins, Models, Molecular, Natronobacterium, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Folding, Sensory Rhodopsins

Journal Title

J Biomol NMR

Conference Name

Journal ISSN

0925-2738
1573-5001

Volume Title

61

Publisher

Springer Science and Business Media LLC
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/K01983X/1)
Biotechnology and Biological Sciences Research Council (BB/E013228/1)
Biotechnology and Biological Sciences Research Council (BB/G011915/1)
This work was supported by the Biotechnology and Biological Sciences Research Council BBSRC [BB/K01983X/1].