Integral membrane protein structure determination using pseudocontact shifts.

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

doi:10.1007/s10858-015-9899-6

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. Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-015-9899-6) contains supplementary material, which is available to authorized users.

Highlights

Protein structure determination by solution-state NMR spectroscopy typically relies to a substantial extent on NOE distance information (Wuthrich 1989)
To investigate the suitability of lanthanide tag-induced pseudocontact shifts for the structure determination of membrane proteins, four phototactic receptor sensory rhodopsin II (pSRII) mutants were prepared in which single cysteine residues were introduced at different positions around the protein (Fig. 1b)
To assess any influence due to effects related to the local environment, mutation sites were chosen such that the lanthanide tags would reside in positions differing substantially in hydrophobicity; with L56C and I121C residing in extracellular loops, S154C in the cytoplasmic region and V169C in the transmembrane region of the protein

Summary

Introduction

Protein structure determination by solution-state NMR spectroscopy typically relies to a substantial extent on NOE distance information (Wuthrich 1989). Selective protonation of particular groups within a perdeuterated sample, such as the selective methyl-protonation strategies pioneered by Kay and coworkers (Tugarinov et al 2006) and subsequently extended to further methylcontaining amino acids by others (Ayala et al 2009; Godoy-Ruiz et al 2010), allow additional NOEs to be measured. Such approaches may not entirely overcome the existing problems, may not be successful for

Methods

Results

Discussion

Conclusion