How to Investigate Interactions Between Membrane Proteins and Ligands by Solid-State NMR

Lakatos, Andrea; Mörs, Karsten; Glaubitz, Clemens

doi:10.1007/978-1-62703-023-6_5

Cited by 9 publications

(11 citation statements)

References 83 publications

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“…In order to filter just the signal of nucleotides bound to the NBDs of MsbA, we utilized dipolar-based cross-polarization (CP) magnetization transfer from the homogeneous 1 H network to 31 P (ref. 40). In this way, one can discriminate between MsbA-bound from free nucleotides.…”

Section: Resultsmentioning

confidence: 99%

Coupled ATPase-adenylate kinase activity in ABC transporters

et al. 2016

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ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.

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Section: Resultsmentioning

confidence: 99%

Coupled ATPase-adenylate kinase activity in ABC transporters

et al. 2016

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“…Proteoliposomes are concentrated into a dense lipid, protein and buffer pellet that is packed into a small rotor for MAS experiments 52 . We describe here an in-house MAS sample transfer protocol to minimize sample loss (Box 2, Fig.…”

Section: Experimental Designmentioning

confidence: 99%

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

2013

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Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. such an environment is often necessary for achieving native-like structures. sample preparation is the key to this success. Here we present a detailed description of a robust protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. the procedure is demonstrated using two proteins: CrgA (two transmembrane helices) and rv1861 (three transmembrane helices), both from Mycobacterium tuberculosis. the success of this procedure relies on two points. First, for samples for both types of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in ‘complete’ removal of detergent. second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d.

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“…HSQC (hetero-nuclear single quantum coherence) spectrum: This is a two-dimensional spectrum that correlates a proton with a hetero nucleus, usually 13 C and 15 N, and it shows one NMR peak per unique proton attached to the particular hetero-nucleus.…”

Section: Cpmg Relaxation Dispersionmentioning

confidence: 99%

“…C) Formation of the MDa complex between the 20S proteasome and the 11S activation domain which is in slow exchange and has μ M affinity . Peaks from both the free and the bound states of C‐methyl labelled 20S proteasome are observed in the methyl TROSY spectra. Reprinted by permission from Macmillan Publishers: Nature (reference 54), copyright (2007).…”

Section: Introductionmentioning

confidence: 99%

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(S)Pinning down protein interactions by NMR

et al. 2017

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Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions. These types of analyses determine the amounts and proportions of individual constituents that participate in a reaction as well as their rates of reactions and their thermodynamics. Although many different methods are available, there is currently no single method able to quantitatively capture and describe all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates, and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative descriptions of protein interactions. In this review we provide an easy‐access approach to NMR for the non‐NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background and illustrate simple protein–ligand interactions as well as typical strategies for measuring binding constants using NMR spectroscopy. Finally, this review provides examples of caveats of the method as well as the options to improve the outcome of an NMR analysis of a protein interaction reaction.

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How to Investigate Interactions Between Membrane Proteins and Ligands by Solid-State NMR

Cited by 9 publications

References 83 publications

Coupled ATPase-adenylate kinase activity in ABC transporters

Coupled ATPase-adenylate kinase activity in ABC transporters

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

(S)Pinning down protein interactions by NMR

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