Why shall we enrich proteins with specific isotopes? Structural determination through NMR 1D spectra contain structural information.. but is hard to extract Dispersed amides: protein is folded Downfield CH 3 : Protein is folded H  region

Why shall we enrich proteins with specific isotopes? Even 2D spectra can be (and indeed are) very crowded Realistic limit of homonuclear NMR: proteins of amino acids; spectra of larger proteins are too crowded

Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance g/10 7 rad T -1 s -1 MHz (2.3 T magnet) 1 H1/ % H C1/ N N1/ O5/ F1/ Na3/ P1/ Cd1/ Useful nuclei such as 15 N, 13 C are rare

The solution is… The solution is  3D heteronuclear NMR  Isotopic labeling

 Uniform labeling Isotopically labeled proteins can be prepared in E. coli by growing cells in minimal media (e.g. M9) supplemented with appropriate nutrients ( 15 NH 4 Cl, 13 C-glucose) or in labelled rich media. Requirements for heteronuclear NMR: isotope labeling  Residue specific labeling Metabolic pathways can be exploited and appropriate auxotrophic strains of E. coli can also be used for selective labeling: e.g. use acetate instead of glucose and obtain selective labeling of certain side chain CH 3

 Labeling in eukaryotic organisms Eukaryotic proteins which are inefficiently expressed in bacteria, due to problems related to disulphide bond formation and folding, can be efficiently expressed and labelled in yeast strains (P. pastoris) Requirements for heteronuclear NMR: isotope labeling  Deuterium labeling For large proteins deuterium labeling provides simplified spectra for the remaining 1 H nuclei and has useful effects on relaxation properties of attached or adjacent atoms (fast relaxation=broad NMR lines)

A standard protocol for isotope labeling O/N Inoculum in unlabeled medium Massive culture in labeled medium Induction + IPTG Harvesting

From protein purification to check folding  Protein isolation and purification Protein isolation and purification will follow the standard procedure which has been set up for unlabelled protein Protein folding can be checked by 1 H NMR and 1 H- 15 N HSQC spectra.  Check folding

How to optimize protein expression?  Choice of culture medium Two main types of culture media can be tested for labeling:  Ready-to-use media like algae or bacteria hydrolysate  Minimal media added with 15 N nitrogen source or/and 13 C carbon source

Minimal media  Minimal media are composed in the lab and are made of nutrients like C and N source, salts, buffering substances, traces elements and vitamins. Carbon source Carbon source can be glucose (the best as gives highest yiels), glycerol, acetate, succinate, methanol, Etc. In case of 13 C labeling the concentration of carbon source can be reduced with respect to unlabelled culture, to reduce costs!!! Checks must be performed before labelling! Nitrogen source Nitrogen source can be NH 4 Cl or (NH 4 ) 2 SO 4 In case of 15 N labeling the concentration of nitrogen source can be reduced with respect to unlabeled culture, to reduce costs!!! Checks must be performed before labeling!

Minimal media  Minimal media are composed in the lab and are made of nutrients like C and N source, salts, buffering substances, traces elements and vitamins. Salts Salts are NaCl/KCl, MgSO 4, CaCl 2 Buffer Buffer usually is phosphate, pH 7.5 Trace elements Trace elements is constituted by a mixtures of metal ions, like Co 2+, Cu 2+, Zn 2+, Mn 2+, Fe 2+ Vitamins Vitamins are thiamine, biotin, folic acid, niacinamide, pantothenic acid, pyridoxal, riboflavin

Ready-to-use media  These media are usually sterile and in the correct dilution They can be used for massive culture in the same way as unlabeled, rich media like LB or 2 x YT.

Comparison between minimal and ready-to-use media Bacterial growth is usually higher in ready-to-use media than in minimal media.

Comparison between minimal and ready-to-use media  But protein expression? It must be tested, case by case, through expression tests :

Strategies to improve protein expression An example:  Grow cell mass on unlabeled rich media allowing rapid growth to high cell density.  Exchange the cell into a labeled medium at higher cell densities optimized for maximal protein expression Marley J et al. J. Biomol. NMR 2001, 20, 71-75

Strategies to improve protein expression In practice: Cells are grown in rich unlabeled medium. When OD 600 = 0.7 cells are harvested, washed with M9 salt solution, w.o. N and C source and resuspended in labeled media at a higher cell concentration. Protein expression is induced after 1 hour by addition of IPTG.

The need of deuteration Why is necessary to enrich the protein with 2 H? Deuteration reduces the relaxation rates of NMR-active nuclei,in particular 13 C It improves the resolution and sensitivity of NMR experiments

Which is the ideal level of deuteration? It depends from the size of the protein In general  for  c  up to 12 ns (20 KDa) 13 C/ 15 N labeling  for  c  up to 18 ns (35 KDa) 13 C/ 15 N labeling and fractional deuteration  for  c above 18 ns 13 C/ 15 N labeling – selective protonation and background deuteration It depends from the type of NMR experiments

The problem to express a deuterated protein Incorporation of 2 H reduces growth rate of organisms (up to 50%) and decreases protein production Deuterium labeling requires different conditions with respect to 13 C and 15 N enrichment and could require bacteria adaptation

Fractional deuteration Random fractional deuteration can be obtained up to a level of 70-75%, in a media with 85% D 2 O with protonated glucose, without bacteria adaptation O/N culture unlabeled Expressing culture labeled >20 h Preinduction culture labeled 2-6 hours OD 600 = As for 13 C- 15 N labelling all the conditions (strain, glucose conc. time of induction, etc.) must be optimized for each protein!!

Deuterium incorporation Fractional deuteration of recombinant proteins determined using mass spectroscopy. ( ) deuteration with [ 2 H] 2 O only. ( ) deuteration with [ 2 H] 2 O and perdeuterated glucose. O’Connell et al. Anal.Biochem. 1998, 265,

Perdeuteration Perdeuteration require a gradual adaptation of  Perdeuteration require a gradual adaptation of bacteria to increasing concentration of D 2 O. Bacterial strains must be accurately selected in order  Bacterial strains must be accurately selected in order to choose that which better acclimates to D 2 O media. For each strain one or more colony must be selected  For each strain one or more colony must be selected which better survives in high level of D 2 O concetration

A protocol for bacteria adaptation to deuterated medium O/N Inoculum in unlabelled medium 40% D 2 O Massive culture 99% D 2 O 60% D 2 O80% D 2 O99 % D 2 O Glycerol stock 40% D 2 O Glycerol stock 60% D 2 O Glycerol stock 80% D 2 O Glycerol stock 99% D 2 O

Is it possible to avoid the adaptation phase? Wüthrich lab has experimented a culture minimal medium supplemented with deuterated algal hydrolysate which allows us to eliminate cells pre-conditioning Wüthrich K. et al J.Biomol.NMR 2004,29, Composition of the Celtone-supplemented media Basic minimal medium 800 ml H 2 O or D 2 O 100 ml M9 solution 2 ml 1M MgSO 4 1 g NH 4 Cl 1 g D-glucose Vitamin mix and trace elements 10 ml of Vitamin mix 2 ml Trace elements solution Aminoacids supplements 1-3 g deuterate algal lysate (CELTONE) dissolved at 30 mg/ml antibiotics

Is it possible to avoid the adaptation phase? SOME RESULTS Wüthrich K. et al J.Biomol.NMR 2004,29, Medium compositionDeuterationAdvantage/disadvantages Minimal medium on 60-92%no N-H/N-D exchange problems Glucose-d + Celtone-dintermediate deuteration can be achieved in H 2 O Minimal medium on 95-97%high deuteration Glucose-d + Celtone-d in D 2 O

Backbone HN Side-chians

Specific labeling Labeling of a protein can be easily achieved on specific residues with 2 strategies:  In a minimal medium containing unlabelled glucose and complemented with the labelled aminoacids. A mixture of the other unlabelled a.a. can be added to prevent any conversion of the labelled ones  In a complete labelled medium, containing great amount of all unlabelled aminoacids except those which are expected to be Labelled (reverse labelling)

Specific labelling: the main problem The most important problem encountered is the metabolic conversion of the labeled aminoacids which might occur during anabolism and/or catabolism.  Use an auxotrophic strain.  Use a prototrophic strain with high concentration of aminoacids to inhibit some metabolic pathways. An example: Labeling of a protein with 13 C 15 N Lys can be performed in unlabeled media with high level of 13 C 15 N Lys to prevent lysine biosinthesis from aspartate conversion. How to prevent this? However, if complete control over the incorporation of amino acids is required, then cell-free methods must be used.cell-free methods

Specific labeling for assignment of 13 C and 1 H methyl from Ile, Leu, Val Full deuteration precludes the use of NOEs for structure determination.  Full deuteration precludes the use of NOEs for structure determination. How to overcome the problem? Reintroduction of protons by using labeled amino acids Reintroduction of protons by using methyl selectivelly protonated metabolic precursors of aliphatic amino acids

The basic strategy of the SAIL approach is to prepare amino acids with the following features: Labelling of six-membered aromatic rings by alternating 12 C- 2 H and 13 C- 1 H moieties Stereo-selective replacement of one 1 H in methylene groups by 2 H. Replacement of two 1 H in each methyl group by 2 H. Stereo-selective modification of the prochiral methyl groups of Leu and Val such that one methyl is 12 C( 2 H) 3 and the other is 13 C 1 H( 2 H) 2. The 20 protein-component SAIL amino acids are prepared based on these design concepts by chemical and enzymatic syntheses. SAIL - Stereo-Array Isotope Labelling

The production of SAIL proteins involves cell-free expression system. This approach indeed minimize metabolic scrambing effects and produces high incorporation rate of the added SAIL amino acid into the target protein.

Specific protonation at ring carbons of Phe, Tyr, and Trp on deuterated proteins NOEs involving aromatic protons are an important source of distance restraints in the structure calculation of perdeuterated proteins  NOEs involving aromatic protons are an important source of distance restraints in the structure calculation of perdeuterated proteins A selective reverse labeling of Phe, Tyr and Trp has been performed in perdeuterated proteins, using shikimic acid, a precursor of the aromatic rings. In this way the aromatic rings of the aminoacids are partially protonated (50%) Rajesh S. et al. J.Biomol.NMR 2003, 27, 81-86

Specific protonation at ring carbons of Phe, Tyr, and Trp on deuterated proteins

The aromatic rings of the aminoacids are partially protonated by using shikimic acid (40-56%) Higher level of protonation are observed in E.coli strains overexpressing a membrane bound transporter of shikimate Complete protonation can be achieved using an auxotrophic strain defective in shikimate production

To obtain CH 3 in perdeuterated protein sample: α-Ketoacid Precursors for Biosynthetic Labeling of Methyl Sites [ 1 H, 13 C]-labeled pyruvate as the main carbon source in D 2 O- based minimal-media expression of proteins results in high levels of proton incorporation in methyl positions of Ala, Ile(γ2 only), Leu, and Val in an otherwise highly deuterated protein. An example of Site-specific labelling

A bacterial protein expression system with 13 C, 1 H pyruvate as the sole carbon source in D 2 O media

Unfortunately, because the protons of the methyl group of pyruvate exchange with solvent, proteins are produced with all four of the possible methyl isotopomers ( 13 CH 3, 13 CH 2 D, 13 CHD 2, and 13 CD 3 ).

IVL - Ile, Val and Leu side-chain methyl groups The IVL labelling scheme produces protein which is uniformly 2 H, 13 C, 15 N-labelled, except for the Ile, Val and Leu side-chains which are labelled as follows:

The protein is produced by expression from bacteria which are grown on minimal medium in D 2 O using 13 C, 2 H-glucose as the main carbon source and 15 NH 4 Cl as the nitrogen source. One hour prior to induction α-ketobutyrate and α-keto-isovalerate (labelled as shown below) are added to the growth medium and lead to the desired labelling of the Ile and the Val and Leu residues, respectively.

SEGMENTAL LABELLING Protein splicing is a posttranslational process in which internal segments (inteins) catalyze their own excision from the precursor proteins with consequent formation of a native peptide bond between two flanking external regions (exteins). Up to now more than three hundred inteins have been identified (see and many of them were extensively characterized. Their self-splicing properties were used to develop very convenient tools for protein engineering. There are two methods based on intein properties that have been used for segmental isotope labeling of proteins: Expressed Protein Ligation (EPL) and Protein Trans-Splicing (PTS).

SEGMENTAL LABELLING

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