NMR sample preparation of membrane proteins in ...nmrresource.ucsd.edu/probetechnology/SamplePreparationMASExperiments.pdfMarassi et al. Methods (2011) Highly stable samples are required
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NMR sample preparation of membrane
proteins in phospholipid bilayers
Sang Ho Park
University of California, San Diego
Structure determination of membrane proteins by solid-state NMR spectroscopy.
Step 1 Prepare a sample of uniformly 13C/15N labeled
proteoliposomes
Step 2
Step 3
Step 4
Resolve individual signals with MAS solid-state
NMR experiments
Assign each signal to
a specific residue
Measure orientation-dependent
frequencies for each residue
Step 5 Calculate three-dimensional
structure
Marassi et al. Methods (2011)
Highly stable samples are required to perform structural studies on membrane proteins by solid-state NMR spectroscopy.
General procedure from bacterial expression to
reconstitution of membrane proteins into lipid bilayers.
By using an efficient protocol, it takes 3 days from the bacterial culture to the final NMR sample of membrane proteins: day 1,
growth of E. coli culture and preparation of IBs; day 2, purification and refolding; day 3, preparation of CXCR1 proteoliposomes
and the final NMR sample. Every step needs optimization for each membrane proteins.
Park et al. BBA (2012)
Efficient expression can be achieved using E. Coli codon
optimized genes.
• Genes are clustered by using factorial correspondence analysis into three classes. Class I contains genes involved in most
metabolic processes. Class II genes correspond to genes highly and continuously expressed during exponential growth. Class
III genes are implicated in horizontal transfer of DNA. One can see that the distribution of codons in class III genes is more or
less even, whereas it is extremely biased in class II genes (in particular, codons terminated in A are selected against).
• Replacing codons that are rarely found in highly expressed E. coli genes with more favourable codons throughout the whole
gene. Codons that have been associated with translation problems in E. coli are marked in red box.
Expression optimized host strains are used for
overexpression of target proteins
• C41(DE3) and C43(DE3) cells for toxic protein expression
– Dumon-Seignovert et al. Protein Expr Purif (2004)
• Non-DE3 BL21 cells for expression with mild lac or tac promoter
• Rare codon optimized host cells
– BL21 (DE3) CodonPlus-RIPL
– BL21 (DE3) CodonPlus-RIL
– BL21 (DE3) CodonPlus-RP
– Rosetta or Rosetta (DE3)
– BL21 CodonPlus-RIL
– BL21 CodonPlus-RP
If the E coli codon optimized genes are not available, rare codon optimized host cells can be used for expression of rare codon
containing genes.
High level expression of fusion protein
Common fusion partners for bacterial expression of membrane proteins• KSI: Ketosteroid Isomerase
• GST: Glutathione S-transferase
• TrpΔLE: Trp-LE leader sequence
• Bcl-XL: B-cell lymphoma-extra large
• MBP: Maltose Binding Protein
pGEX2a-CXCR1-His
6119 bp
APr
GST
CXCR1 Rezeptor
lac I
His tag
Ptac
ORI
rrnBT1T2
Thrombin cleavage site
CXCR1 receptor
TrpΔLE
p7
BL21(DE3) BL21
p7 CXCR1
No single solution exists for successful production of all recombinant membrane proteins. Instead, it is beneficial to have
access to a wide range of expression tools.
Purification of Vpu from HIV-1
1. Supernatant after cell lysis
2. Inclusion bodies
3. Ni-NTA purified fusion protein
4. CNBr cleaved protein
5. HPLC purified protein
SDS-PAGE HPLC
Fusion partner
Fusion partner
Acquiring high-quality NMR spectra demands the production of samples with high levels of purity and excellent homogeneity
throughout the sample.
Size exclusion chromatography of human chemokine
receptor CXCR1
Pure monomeric receptor
The CXCR1 samples purified by nickel affinity chromatography that contain a small amount of higher oligomers must be
immediately loaded onto the size exclusion chromatography, since trace amount of oligomers (and/or other impurities)
apparently facilitate the formation of larger amounts of higher oligomers.
Reconstitution of CXCR1
1) Mixed micelle solutions were prepared by dissolving the DMPC
powder with 0.5% SDS in 20 mM HEPES buffer pH 7.3, 50 mM
NaCl with a final lipid concentration of 10 mg/ml.
2) Purified monomeric CXCR1 was added to the mixed micelle
solution at a protein-to-lipid ratio of 1:10 or 1:5 (w/w).
3) Incubated for 1 h at room temperature.
4) Detergents were removed to obtain highly pure proteoliposomes.
Detergent removal
• Adsorption to BioBeads.
– fast and effective for low-CMC detergents
– lipid loss due to non-specific adsorption
• Complexation to methyl-β-cyclodextrin.
– Applicable to broad range of detergents
– Reproducible without loss of lipids
• Co-precipitation with potassium chloride.
– Only Suitable for detergents having sulfate head groups (e.g. SDS)
– Reproducible without loss of lipids
Lipid analysis using HPLC-ELSD system
Before reconstitution After reconstitution
retention (ml)
HPLC with Evaporative Light Scattering Detector (ELSD)
It is important for sample quality control to make sure that there are no detergents left after reconstitution in order to obtain
highly pure proteoliposomes for NMR experiments.
Sample preparation for MAS NMR experiments
1) Concentrate proteoliposomes using ultracentrifugation.– 145,000 g, 2 hr – overnight, 15oC.
– Spinning time needs optimization for RA solid-state NMR experiments.
2) Transfer proteoliposomes to MAS rotor using Microman® (Gilson).– Concentrated proteoliposomes are highly viscous.
– Microman® M50 and M10 are used for 3.2 mm rotor and 1.3 mm rotor, respectively.
– Pack the sample evenly by using a centrifuge to ensure that the rotor is properly balanced during MAS.
1) Place an insert and seal the rotor with a rotor cap.– An insert improves shimming and RF in homogeneity.
– Check the integrity of the cap before use and make sure it fits snugly on the rotor.
Rotors and tools for MAS sample preparations
3.2 mm rotor up to 24 kHz spinning rate
for moderate spinning MAS experiments
1.3 mm rotor up to 67 kHz spinning rate
for fast spinning MAS experiments
Microman® M50 and CP50
for 3.2 mm rotor packing
Microman® M10 and CP10
for 1.3 mm rotor packing
http://www.theresonance.com/2014/categories/material-science/nmr-tutorial-mas-rotor-filling
https://www.youtube.com/watch?v=K_0yPPmFYdE#t=118
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