Structure determination of α-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins

Abstract

The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein-protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR

“…Various systems for isotope labeling of membrane proteins for solution and ssNMR have been extensively discussed in the literature (Gautier, 2014;Goncalves, Ahuja, Erfani, Eilers, & Smith, 2010;Kim, Howell, Van Horn, Jeon, & Sanders, 2009;Maslennikov & Choe, 2013;(Kim et al, 2009;Takahashi & Shimada, 2010). In our view, these conditions are satisfied by the expression and isotopic labeling in the methylotrophic yeast Pichia pastoris, which will be described in this chapter using two examples, fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1 (hAQP1) (Emami, Fan, Munro, Ladizhansky, & Brown, 2013;Fan, Shi, Ladizhansky, & Brown, 2011).…”

Application of Solution NMR to Structural Studies on α-Helical Integral Membrane Proteins

“…Although membrane protein expression is important, this topic will not be discussed any further in this review. Instead, several excellent reviews are directly available to the reader [ 20 , 22 ].…”

“…Meanwhile, when a low-resolution structure of protein complex composed of several proteins was obtained by EM spectroscopy, the quality of whole protein complex structure can be improved by fitting of high-resolution structure of unit protein determined by NMR spectroscopy. So far, many excellent reviews have addressed MP expression, NMR techniques, and the progress of structural studies using NMR [ 19 , 20 , 21 , 22 , 23 , 24 ]. This review will briefly provide guidelines on what needs to be done to determine the MP structure by solution NMR analysis, and discuss some details on membrane-mimetic media according to the analysis of the structures deposited in PDB.…”

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR

“…Structures solved using this technique include those of the microbial photosensor Anabaena sensory rhodopsin [22], chemokine receptor CXCR1 [23], Influenza A M2 channel [24], transmembrane protein CrgA from Mycobacterium tuberculosis [25], and outer membrane protein G [26]. These successes can be extended to other eukaryotic membrane proteins, some of which are biomedically relevant, but harder to express and reconstitute for SSNMR in a structurally homogeneous form [6,[27][28][29][30][31][32][33].…”