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Isotope labelling
Isotopes are different types of atoms of the same chemical element, each having a different number of neutrons:
126c ,36c "6c
stable isotopes radioactive isotope
Stable isotope labelling - a very powerful tool in NMR studies of proteins: spectral overlap reduction — f spectral resolution — facilitates the study of the structure and dynamics of the proteins and protein complexes
NMR spectroscopy - solution of protein is placed in strong magnetic field; then bombarded with radio waves - hydrogen nuclei generate NMR signals (spectrum) indicating distances between atoms
Obtaining isotopically labelled
proteins
1. Strategies
1. uniformly labelled
2. selectively labelled
3. segmental isotope labelling
2. Approaches
1. in vivo: by expressing the corresponding gene in host organisms, which grow on isotope-enriched media
2. in vitro: cell-free synthesis system
1.1.2. 15N uniformly labelled
The simplest and the cheapest labelling Nitrogen source: 15NH4Cl or (15NH4)2SO4 Applications:
- standard solution-NMR HSQC (heteronuclear single quantum coherence) experiment — spectrum: folded x unfolded protein
- dynamics experiments
- titrations with ligands forming complex
- small proteins (< 150 aa) 1H and 15N backbone assignment using 15N-NOESY and 15N-TOCSY
http://www.protein-nmr.org.uk/spectra.html
15N-HSQC of CKI1RD 11                   10                    9                    8                    7 6		
110		■110
115-		115
3~ 120 125		125
		
		
11                   10                   9                    8                    7 6 C02- 1H (ppm)		
1.1.3. 15N, 13C (double labelling)
• Carbon source: 13C-glucose
• Applications
- a very common form of labelling
- assignment of both the backbone and side-chain 1H, 13C and 15N atoms using triple-resonance spectra (structure determination of proteins ~ 20 kDa)
1.1.4. 15N, 13C, 2H (triple labelling)
• Medium in D2O instead of H2O
• Applications
- mainly used for structural studies of large proteins (20-80 kDa): by deuterating the protein and thus removing most 1H atoms (protons), the relaxation properties are improved — | sensitivity and spectral resolution
1.1.3. 15N, 13C
HNCO
triple resonance experiments (3D)
HN(CA)CO
CBCA(CO)NH / HN(CO)CACB
@—@—@ @—K)—@
(Hp)-<Cp)-(Hp)      (hb)-(ci-(Hp)
CBCANH / HNCACB
^Tj-@-@      @-(cj)-(HyJ
{H P)—4fir—■ h P)    ' h p')—i»—( h p;
http://www.protein-nmr.org.uk/spectra.html
2D spectrum
1.2. Selective isotope labelling 1.2.1. Ile, Val and Leu side-chain methyl groups
To improve structure calculations of large proteins
The IVL labelling produces uniformly 2H,13C,15N-labelled proteins, except for the Val, Leu, and Ile side-chains which are labelled as follows:
N H
1JCSH— 13C'H— l3'
O
15M I
NH
.0
0
(1H-y methyl)-Val
,3C?H    13C*H     13CaH— 13C
(1H-5 methyl)-Leu
(1H-51 methyl)-Ile
The protein is produced by expression from bacteria which are grown on MM in D2O using 13C,2H-glucose and 15NH4Cl. One hour prior to induction, labelled a-ketobutyrate and a-ketoisovalerate are added.
[3-2H] a-ketoisovalerate —► Val, Leu
[3,3-2H2] a-ketobutyrate —► Ile
1.2. Selective isotope labelling
Chemical structures of metabolites Involved In selective Isotope labeling strategies.		
Labeling agent	Chemical structure	Incorporated as
[3-2H] a-ketoisovalerate	*CH3-*CD-*CO-*COO" *CH3	t1 H-B methyl)-Leu t1H-y methyl)-Val
a-ketobutyrate	*CH3-*CD2-*CO-*COO"	(1H-B1 methyl)-lie
[e-iaC] L-phenylalanine	COO" CH-CH2^) NH3+	[E-iaG] Phe
[2-13C] or [1:3-13C2] glycerol	CHa-OH *CH2-OH *CH-OH   or CH-OH CH2-OH *CH2-OH	12C-13C-12C pattern
13C pyruvate	*CH3-*CO-*COO^	(1H-S methyl)-Leu (1H-y methyl)-Val methyO-lle (1H-p methyl)-Ala
[3-13C] pyruvate	*CH3-CO-COO^	[13C-B methyl)-Leu Í^C-ř methyl)-Val (13C-t2 methyl)-lle (12-c-IJ methyl)-Ala
* lndicates positions |abe||ed by 13C (Goto & Kay, 2000)
1.2.2. 1,3-13C- and 2-13C-glycerol
Source: 15NH4CI and either 1,3-13C- or 2-13C-labelled glycerol
*CH2-OH CH-OH *CH2-OH
Applications
- to measure relaxation rates —► to study internal dynamics of side-chains within proteins (Thioredoxin: LeMaster and Kushlan, 1996)
- for protein structure determination in solid-state MAS (magic-angle-spinning) NMR (Castellani et al., 2002)
blue: 1,3-13C glycerol labelling red: 2-13C glycerol labelling
CHa-OH 'CH-OH CH2-OH
NH2 0H	Gin R-00€^X£f° Glu            :           1 xoh Pro L-^rNH=
Ser               K   N ■	Asn ^° Asp R^ffHZKlf Mel            T T ^nH Thr                   (ho) NH2 un
Tyr                        NH2 OH	Lys o NH2 0H
Trp	.0
	.0 0 • NH2 0H
	
http://www.protein-nmr.org.uk/labelling.html#aaspecific
1.2.3. Selective isotope labelling -
other strategies
• Expression from bacteria which are grown on MM supplemented with small amounts of 15NH4Cl and 13C-glucose as well as labelled and unlabelled amino acids.
• Reverse labelling - The protein is produced by expression from bacteria which are grown on MM supplemented with 15NH4Cl and 13C-glucose as well as unlabelled amino acids.
This suppresses the labelling of these amino acids and only those which have not been added unlabelled will be synthesised by the bacteria using the 13C-glucose as the carbon source.
1.2.4. SAIL - stereo-array isotope labelling (Kainosho et al., Nature 2006)
SAIL-labelled proteins are prepared using cell-free technology.
The amino acids used for the protein production are prepared using chemical and enzymatic syntheses.
Their labelling is guided by the following principles:
- in each methylene group, one of the 1H atoms is stereo-selectively replaced by a 2H atom;
- in each methyl group, two of the 1H atoms are replaced by 2H atoms;
- the prochiral methyl groups of Leu and Val are stereo-selectively 12C2H3 and 13C1H2H2 labelled;
- six-membered aromatic rings have alternating 12C2H and 13C1H moieties.
For the structure calculation of large proteins For the investigation of side-chain motions Very expensive
20 SAIL amino acids
http://www.sailnmr.org/wiki/index.php/File:SailAminoAcids.jpg
1.3. Segmental isotope labelling
Applications
To investigate interdomain interactions within multidomain proteins
To study conformational changes after ligand binding To help resonance assignment of large proteins To facilitate protein structure determination
Approaches
1. Native chemical ligation
2. Expressed protein ligation
3. Protein trans-splicing
(Skrisovska et al., 2010)
1.3.1. Native chemical ligation
• Based on an interaction of two synthetic peptides (~ 50 aa), one containing C-terminal a-thioester and the other N-terminal a-cysteine — formation of native peptide bond
• one peptide can be labelled and one unlabelled
Native Chemical Ligation NCL
(Skrisovska et al., 2010)
1.3.2. Expressed protein ligation
• = intein-mediated protein ligation
• Based on native chemical ligation and inteins properties
• Involves recombinant expression of one or both protein fragments [IMPACT™ system (NEB): set of bacterial vectors allowing recombinant
___I     I ■____r _     I   ■    r______I Expressed Protein
production of protein fragments Ligationepl
with a-thioesters and a-cysteines
(Skrisovska et al., 2010)
1.3.3. Protein trans-splicing
• Based on protein trans-splicing process = Inteins are fragmented in two inactive parts. After their association, they reconstitute into active intein which performs a splicing reaction resulting in ligation of their fusion protein fragments.
Protein Trans-Splicing Protein Trans-Splicing PTS in vivo
PTS in vitro
1.3.3. Protein trans-splicing in multidomain
protein
/- N-term
Intein I N-term
Plasmid I
] lntein 1 'ILb-MiJI lntein 11
Plasmid II
lntein II C-term
Plasmid III
1. Express separately using different isotopic labelling
N-temn
2. Split inteins fold into functional inteins
Intein N-term    J Intein C-term J
Intein N-ferrn
Intein C-term
3. N-, middle and C-terminal segments of target protein are joined together covalenily and split from the intein fragments
http://www.protein-nmr.org.uk/labelling.html
2. Protein expression systems
2.1. in vivo
• Escherichia coli
• Methylotrophic yeast Pichia pastoris
• Baculovirus expression system (BVES)
• Mammalian cells
• Slime mold
• Hybridoma cells
• Plants
2.2. in vitro
• Cell-free expression system
2.1.1. Escherichia coli
• Advantages
- the most economical system (easy genetic manipulation, easy culturing conditions, rapid population growth, high-level protein production)
• Disadvantages
- unwanted metabolic conversion to other amino acids (scrambling, e.g. 14N Val —► 15N Ala, 15N Leu —► 15N Glu)
- lack of intracellular organelles (Golgi system, endoplasmic reticulum)
- limited number of molecular chaperones
- absence of post-translational modification
i
Some eukaryotic proteins (containing disulfide bonds) cannot be folded correctly and are expressed insolubly as inclusion bodies.
2.1.1. Escherichia coli
Strategies to overcome some limitations: • Scrambling:
- the use of auxotrophic strains and/or suppression of transaminase activity
- inhibition with a 10-fold excess of unlabelled amino acids in medium relative to the labelled aa
• Insolubility:
- I temperature of expression
- adding highly soluble tags (GST, TrxA, ...) to target proteins
- the use of other expression systems:
» Corynebacterium glutamicum - expression of soluble Streptoverticillium mobaraense transglutaminase (Shinagawa et al., 2005)
» Brevibacillus choshinensis (Tanio et al., 2009; Udaka & Yamada, 1993)
» other non-bacterial systems
2.1.1. Escherichia coli
Expression of CKI1RD
1. TB medium, 37CC ® 28CC, 3 h
2. M9 minimal medium, 37°C ® 28°C, 15 h
3. M9 minimal medium, 37°C ® 25°C, 15 h
4. M9 minimal medium, 37°C ® 28°C, 3 h
5. M9 minimal medium, 29°C ® 22°C, 3 h
6. M9 minimal medium, 22°C ® 22°C, 3 h
Purification of 1 L of bacterial culture: M9 medium ® 7-10 mg of protein
TB medium ® 20-25 mg of protein
2.1.2. Pichia pastoris
• Advantages
- easy genetic manipulation
- yeast grows to high cell density
- high yield of secreted protein (100-500 mg/L when using a
fermenter)
- capable of post-translational modifications (glycosylation, proteolytic processing, disulfide bond formation)
- stable isotope labelling of secreted proteins
• Disadvantages
- inability of some post-translational modifications: prolyl hydroxylation, certain types of phosphorylation, high mannose glycosylation (N- and O-linked glycosylation patterns are different from higher eukaryotes)
- P. pastoris uses alcohol oxidase 1 promoter induced by methanol; due to toxicity, methanol must be strictly controlled during cultivation — Kluyveromyces lactis (promoter LAC4 induced by galactose)
2.1.3. Baculovirus expression system
• For the expression of mammalian proteins such as kinases and membrane proteins.
• Based on the infection of insect cells (Sf9 cells) with a recombinant baculovirus carrying target gene and the subsequent expression of target protein by the insect cells.
• Disadvantages
- insect cells don't grow in minimal medium — expensive labelled rich media
- slow growth
- low yields
- eukaryotic cells cannot survive in deuterium oxide media (toxic for them) — combination of amino acid-type selective 15N labelling and several basic triple resonance experiments
2.1.4. Mammalian cells: Chinese hamster ovary (CHO) cells, HEK 293 cells
• For production of mammalian proteins
• Advantages
- Contain a mammalian N-glycosylation system — production of "authentic" mammalian glycoproteins
- Amino acids are directly incorporated from the medium into the expressed protein — no scrambling of isotope labels
• Disadvantages
- Slow growth
- Low protein yield
- Cells cannot grow on the isotopically enriched minimal media, mostly require serum — uniform isotope labelling is expensive — serum free medium supplemented with labelled amino acids purified from the bacterial or algal hydrolysate (Hansen et al.,
1992)
- eukaryotic cells cannot survive in deuterium oxide media (toxic for them) — combination of amino acid-type selective 15N labelling and several basic triple resonance experiments
2.1.5. Slime mold Dictyostelium discoideum
• Promising eukaryotic expression system
• Advantages
- Rapid cell growth
- Simple media
- Good yields (~ 9 mg from 10 g 13C glycerol (Cubeddu et al., 2000)
2.1.7. Hybridoma cells
• For production of uniformly 15N/13C labelled antibodies using serum free media
• Yields 20-40 mg/l cell culture
2.1.8. Plants
• Advantages
- all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins
- Nitrogen source: K15NO3,15NH415NO3
- Carbon source: 13C sucrose
Uniformly 15N-labelled (>98%) potato plants (Ippel et al., 2004)
Hydroponic system (K15NO3)    Potato tuber lysate       Purified protease inhibitor
PSPI:6.5
2.2. Cell-free expression system
• In vitro protein expression:
DNA or mRNA for the target protein is added to the cell lysate (derived from E. coli or wheat germ) containing all the cellular components for protein expression (transcription and translation machinery) along with 20 amino acids, nucleoside triphosphates, several enzymes as well as buffers, salts, etc.
• Advantages
- Can incorporate variety of reagents (e.g. detergents, protease inhibitors, chaperones, ligands) — may facilitate protein synthesis, folding, post-translational protein stability — useful for producing cytotoxic, integral membrane proteins, proteins containing multiple disulfide bonds (elimination of cellular transport and toxicity)
2.2. Cell-free expression system
• Advantages
- The target protein is the only protein synthesized and labelled
- Efficient technique for selecting labelling of certain aa and for specific aa position
- Incorporation of non-natural aa (Fluoro-tryptophan [Neerathilingam et. al., 2005], L-3,4-dihydroxyphenylalanine [DOPA; Ozawa et al., 2005])
- Reduces reaction volumes (pl-ml), quantities of expensive and unusual labelled aa, isotopic scrambling (transaminase activities are suppressed)
- Commercial cell-free expression kits (expensive for large scale production - 1 ml ~ $350)
• Disadvantages
- Expensive equipment: Roche RTS Proteomaster reaction device
- Low yield of protein
- Not all proteins are synthesized in vitro
Labelling for X-ray crystallography
• Incorporation of selenomethionine into proteins in place of methionine aids the structure elucidation of proteins using multi-wavelength anomalous diffraction (MAD).
- The incorporation of heavy atoms such as selenium helps solving the phase problem in X-ray crystallography.
• Neutron protein crystallography provides a powerful
complement to X-ray crystallography by enabling key hydrogen atoms to be located in biological structures that cannot be seen by X-ray analysis alone.
- The availability of a fully deuterated protein eliminates the hydrogen incoherent scattering contribution to the background and brings further ~10-fold improvements in the signal to noise ratios.
- The neutron Laue diffractometer LADI, run jointly by EMBL and ILL at the ILL high flux reactor in Grenoble, is a dedicated facility for neutron protein crystallography at high-resolution (1.5 A).
Literature
• Staunton et al. (2006): Cell-free expression and selective isotope labelling in protein nMr. Magn. Reson. Chem. 44: S2-S9.
• Skrisovska et al. (2010): Recent advances in segmental isotope labeling of proteins: nMr applications to large proteins and glycoproteins. J. Biomol. nMr 46: 51-65.
• Takahashi & Shimada (2010): Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells. J. Biomol. NMR 46: 3-10.
• Goto & Kay (2000): New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr. Opin. Struct. Biol. 10: 585-592.