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).&#160;This article was supported by iNEXT-Discovery (project number 871037).

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.
<ol>
	<li>Use a single colony of E. coli BL 21 Star (DE3) containing the pET11a&#916;ssYaeT plasmid enc...

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&#160;systems devoid of membranes. In this article, an experimental approach is presented that allows to obtain atomistic i...

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 &#62;1,000 MHz, fast magic angle spinning frequencies &#62;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.

<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium molibdate</td>
			<td>Merck</td>
			<td>277908</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium-15N Chloride</td>
			<td>Cortecnet</td>
			<td>CN80P50</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td></td>
		</tr>
		<tr>
			<td>AMUpol</td>
			<td>Cortecnet</td>
			<td>C010P005</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>EMD Millipore Corp</td>
			<td>70746-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Merck</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Sigma</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium dichloride</td>
			<td>Merck</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline chloride</td>
			<td>Sigma</td>
			<td>C-1879</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt chloride</td>
			<td>Merck</td>
			<td>449776</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper sulphate</td>
			<td>Merck</td>
			<td>C1297</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Biotin</td>
			<td>Merck</td>
			<td>8512090025</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium Oxide</td>
			<td>Cortecnet</td>
			<td>CD5251P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck</td>
			<td>D9170</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma</td>
			<td>F-7876</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose 13C + 2H</td>
			<td>Cortecnet</td>
			<td>CCD860P50</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Honeywell</td>
			<td>G7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol (12C3, 99.95% D8, 98%)</td>
			<td>Eurisotope</td>
			<td>CDLM-8660-PK</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol (non-enriched)</td>
			<td>Honeywell</td>
			<td>G7757-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Roth Carl</td>
			<td>NR.0037.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron sulphate</td>
			<td>Merck</td>
			<td>307718</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Thermofisher</td>
			<td>R0392</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny Broth</td>
			<td>Merck</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride - hexahydrate</td>
			<td>Fluka</td>
			<td>63064</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulphate</td>
			<td>Merck</td>
			<td>M5921</td>
			<td></td>
		</tr>
		<tr>
			<td>monopotassium phosphate</td>
			<td>Merck</td>
			<td>1051080050</td>
			<td></td>
		</tr>
		<tr>
			<td>Myoinositol</td>
			<td>Sigma</td>
			<td>I-5125</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Dodecyl-B-D-maltoside</td>
			<td>Acros Organics</td>
			<td>3293702509</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethyldodecylamine N-oxide</td>
			<td>Merck</td>
			<td>40236</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicatinamide</td>
			<td>Sigma</td>
			<td>N-3376</td>
			<td></td>
		</tr>
		<tr>
			<td>Panthotenic acid</td>
			<td>Sigma</td>
			<td>21210-25G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>protease inhibitor</td>
			<td>Sigma</td>
			<td>P8849</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal-HCl</td>
			<td>Sigma Aldrich</td>
			<td>P9130</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Aldrich</td>
			<td>R170-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck</td>
			<td>K51107104914</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phospahte - monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>1,06,34,61,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Thermo-scientific</td>
			<td>28365</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>1,06,49,81,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Life Science</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine-HCl</td>
			<td>Merck</td>
			<td>5871</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma Aldrich</td>
			<td>10,70,89,76,001</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc chloride</td>
			<td>Merck</td>
			<td>208086</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL21 DE3*</td>
			<td>New England Biolabs</td>
			<td>C2527</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Ultra-tubes</td>
			<td>Beckman Coulter</td>
			<td>357448</td>
			<td></td>
		</tr>
		<tr>
			<td>30 kDa centrifugal filter</td>
			<td>Amicon</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm sapphire DNP rotor with caps</td>
			<td>Cortecnet</td>
			<td>H13861</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm teflon insert</td>
			<td>Cortecnet</td>
			<td>B6628</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm sample packer/unpacker</td>
			<td>Cortecnet</td>
			<td>B6988</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm Regular Wall MAS Rotor</td>
			<td>Cortecnet</td>
			<td>HZ16913</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm Regular Wall MAS rotor</td>
			<td>Cortecnet</td>
			<td>HZ09244</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool Kit for 3.2 mm Thin Wall rotor</td>
			<td>Cortecnet</td>
			<td>B136904</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 mm MAS rotor + caps</td>
			<td>Cortecnet</td>
			<td>HZ14752</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 mm filling tool</td>
			<td>Cortecnet</td>
			<td>HZ14714</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 mm sample packer</td>
			<td>Cortecnet</td>
			<td>HZ14716</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 mm cap remover</td>
			<td>Cortecnet</td>
			<td>HZ14706</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 mm cap set tool</td>
			<td>Cortecnet</td>
			<td>HZ14744</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing 12-14 kDa</td>
			<td>Spectra/Por</td>
			<td>132703</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpie - Black</td>
			<td>Merck</td>
			<td>HS15094</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution studies of proteins in natural membranes by solid-state nmr

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.

Figure 2&#160;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...

Read this Scientific Article Protocol about High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR at JoVE.com

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

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.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...