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

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 proteins^2,3,4,5,6,7 and has long been used to study membrane proteins, including membrane embedded protein pumps⁸, channels

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.

1. Use a single colony of E. coli BL 21 Star (DE3) containing the pET11aΔssYaeT plasmid encoding for E. coli BamA-P4P5 to inoculate 50 mL of Lysogeny broth supplemented with 50 µg/L of ampicillin.
2. Grow the culture at ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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