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Abstract
Introduction
Protocol
Results
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Chemistry

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Published: March 03, 2021
doi:
10.3791/62197
, , , ,
1NMR Spectroscopy, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Faculty of Science,Utrecht University

Summary

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Abstract

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

Introduction

Structural and motional studies of membrane proteins in physiologically relevant environments pose a challenge to traditional structural biology techniques1. Modern solid-state nuclear magnetic resonance spectroscopy (ssNMR) methods offer a unique approach for the characterization of membrane proteins2,3,4,5,6,7 and has long been used to study membrane proteins, including membrane embedded protein pumps8, channels9,10,11, or receptors12,13,14,15. Technical advances such as ultra-high magnetic fields >1,000 MHz, fast magic angle spinning frequencies >100 kHz, and hyperpolarization techniques16 have established ssNMR as a powerful method for the study of membrane proteins in environments of ever-increasing complexity from liposomes to cell membranes and even whole cells. For example, DNP has become a powerful tool for such experiments (see reference17,18,19,20,21,22,23,24,25). More recently, 1H-detected ssNMR offers increasing possibilities to study membrane proteins at high spectral resolution and sensitivity25,26,27,28,29. This work highlights two bacterial membrane proteins that are involved in essential functions, i.e., protein insertion and ion transport. The corresponding proteins, BamA25,30,31,32,33 and KcsA23,27,28,34,35,36,37,38,39 (or chimeric variants thereof10,40) have been examined by ssNMR methods for more than a decade.

A representative protocol for the preparation and ssNMR characterization of bacterially originating membrane proteins is presented here. The different steps of the protocol are shown in Figure 1. First, the expression, isotope-labeling, purification, and membrane-reconstitution of BamA is explained. Then, a general workflow for the characterization of the membrane protein by ssNMR is presented; specifically, the assignment of membrane protein backbones using 1H-detected ssNMR at fast magic angle spinning. Finally, basic setup and acquisition of dynamic nuclear polarization-(DNP)-supported experiments, which significantly boost ssNMR signal sensitivity, are detailed.

Protocol

1. Production of uniformly labeled 2H, 13C, 15N-labeled BamA-P4P5 NOTE: While this protocol requires working with non-pathogenic Gram-negative bacteria, adherence to basic biological safety procedures is a must, namely, wearing safety glasses, lab coats, gloves, and following institutional standard operating procedures for work with microorganisms. Use a single colony of E. coli BL 21 Star (DE3) containing the pET11aΔssYaeT plasmid enc…

Representative Results

Figure 2 shows representative gels for inclusion body purity (Panel A) and refolding of inclusion bodies (Panel B3). Figure 2 confirms the successful purification of 13C,15N-labeled BamA-P4P5. Figure 3A shows a typical 2D 13C-13C spectrum of a well-ordered membrane protein, and Figure 3B shows a typical, high-quality 2D 15…

Discussion

Membrane proteins are key players in the regulation of vital cellular functions both in prokaryotic and eukaryotic organisms; thus, understanding their action mechanisms at atomic levels of resolution is of vital importance. The existing structural biology techniques have pushed scientific understanding of membrane proteins quite far but have heavily relied on experimental data gathered from in vitro systems devoid of membranes. In this article, an experimental approach is presented that allows to obtain atomistic i…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work is part of the research programs ECHO, TOP, TOP-PUNT, VICI, and VIDI with project numbers 723.014.003, 711.018.001, 700.26.121, 700.10.443, and 718.015.00, which are financed by the Dutch Research Council (NWO). This article was supported by iNEXT-Discovery (project number 871037).

Materials

Ammonium molibdate Merck 277908
Ammonium-15N Chloride Cortecnet CN80P50
Ampicillin Sigma Aldrich A9518
AMUpol Cortecnet C010P005
Benzonase EMD Millipore Corp 70746-3
Boric acid Merck B6768
bromophenol blue Sigma B0126
calcium dichloride Merck 499609
Choline chloride Sigma C-1879
Cobalt chloride Merck 449776
Copper sulphate Merck C1297
D-Biotin Merck 8512090025
Deuterium Oxide Cortecnet CD5251P1000
Dimethyl sulfoxide Merck D9170
Ethylenediaminetetraacetic acid Sigma Aldrich L6876
Folic acid Sigma F-7876
Glucose 13C + 2H Cortecnet CCD860P50
Glycerol Honeywell G7757
Glycerol (12C3, 99.95% D8, 98%) Eurisotope CDLM-8660-PK
glycerol (non-enriched) Honeywell G7757-1L
Glycine Sigma Aldrich 50046
Guanidine hydrochloride Roth Carl NR.0037.1
Iron sulphate Merck 307718
isopropyl β-D-1-thiogalactopyranoside Thermofisher R0392
Lysogeny Broth Merck L3022
Lysozyme Sigma Aldrich L6876
Magnesium chloride – hexahydrate Fluka 63064
magnesium sulphate Merck M5921
monopotassium phosphate Merck 1051080050
Myoinositol Sigma I-5125
n-Dodecyl-B-D-maltoside Acros Organics 3293702509
N,N-Dimethyldodecylamine N-oxide Merck 40236
Nicatinamide Sigma N-3376
Panthotenic acid Sigma 21210-25G-F
protease inhibitor Sigma P8849
Pyridoxal-HCl Sigma Aldrich P9130
Riboflavin Aldrich R170-6
Sodium Chloride Merck K51107104914
Sodium dihydrogen phospahte – monohydrate Sigma Aldrich 1,06,34,61,000
Sodium dodecyl sulfate Thermo-scientific 28365
Sodium hydroxide Merck 1,06,49,81,000
Sucrose Sigma Life Science S9378
Thiamine-HCl Merck 5871
Tris-HCl Sigma Aldrich 10,70,89,76,001
Zinc chloride Merck 208086
E.coli BL21 DE3* New England Biolabs C2527
1.5 mL Ultra-tubes Beckman Coulter 357448
30 kDa centrifugal filter Amicon UFC903024
3.2 mm sapphire DNP rotor with caps Cortecnet H13861
3.2 mm teflon insert Cortecnet B6628
3.2 mm sample packer/unpacker Cortecnet B6988
3.2 mm Regular Wall MAS Rotor Cortecnet HZ16913
3.2 mm Regular Wall MAS rotor Cortecnet HZ09244
Tool Kit for 3.2 mm Thin Wall rotor Cortecnet B136904
1.3 mm MAS rotor + caps Cortecnet HZ14752
1.3 mm filling tool Cortecnet HZ14714
1.3 mm sample packer Cortecnet HZ14716
1.3 mm cap remover Cortecnet HZ14706
1.3 mm cap set tool Cortecnet HZ14744
Dialysis tubing 12-14 kDa Spectra/Por 132703
Sharpie – Black Merck HS15094
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Keywords: Membrane ProteinsSolid-state NMRStructureDynamicsNatural Lipid MembranesBamAKcsAIsotope LabelingDNPProton-detected NMR
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Beriashvili, D., Schellevis, R. D., Napoli, F., Weingarth, M., Baldus, M. High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR. J. Vis. Exp. (169), e62197, doi:10.3791/62197 (2021).
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