Solution NMR study of integral membrane proteins.
ABSTRACT Signals between a cell and its environment are often transmitted through membrane proteins; therefore, many membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, are important drug targets. Structural information about membrane proteins remains limited owing to challenges in protein expression, purification and the selection of membrane-mimicking systems that will retain protein structure and function. This review describes recent advances in solution NMR applied to the structural study of integral membrane proteins. The examples herein demonstrate that solution NMR spectroscopy will play a unique role not only in structural analysis, but also drug discovery of membrane proteins.
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ABSTRACT: Bicelles are model membranes generally made of long-chain dimyristoyl-phosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptide since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objective of this work was to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using 31P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2 to 400 mM) and q ratios (0.15 to 2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ≤ 250 mM and especially at q ~1.5-2 since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q>1. Boundaries are presented within which control of the bicelles' q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.Langmuir 05/2014; · 4.38 Impact Factor
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ABSTRACT: Phospholamban (PLB) is a membrane protein that regulates heart muscle relaxation rates via interactions with the sarcoplasmic reticulum Ca2+ ATPase (SERCA). When PLB is phosphorylated or Arg9Cys (R9C) mutated, inhibition of SERCA is relieved. 13C and 15N solid-state NMR spectroscopy is utilized to investigate conformational changes of PLB upon phosphorylation and R9C mutation. 13C=O NMR spectra of the cytoplasmic domain reveal two α-helical structural components with population changes upon phosphorylation and R9C mutation. The appearance of an unstructured component is observed on domain Ib. 15N NMR spectra indicate an increase in backbone dynamics of the cytoplasmic domain. Wild-type PLB (WT-PLB), Ser16 phosphorylated PLB (P-PLB) and R9C mutated PLB (R9C-PLB) all have a very dynamic domain Ib and the transmembrane domain has an immobile component. 15N NMR spectra indicate that the cytoplasmic domain of R9C-PLB adopts an orientation similar to P-PLB and shifts away from the membrane surface. Domain Ib (Leu28) of P-PLB and R9C-PLB loses the alignment. The R9C-PLB adopts a conformation similar to P-PLB with a population shift to a more extended and disordered state. The NMR data suggests the more extended and disorder forms of PLB may relate to inhibition relief.The Journal of Physical Chemistry B 02/2014; · 3.38 Impact Factor
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ABSTRACT: Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [15N-1H]- and [13C-1H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan 15N backbone positions and also resolved signals for 15N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [13C-1H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.Molecular Membrane Biology 04/2014; 31(4):131-140. · 1.73 Impact Factor