Why shall we enrich proteins with specific isotopes?

1. Why shall we enrich proteins with specific isotopes? Structuraldeterminationthrough NMR 1D spectra contain structural information .. but is hard to extract Ha region Dispersed amides: protein is folded Downfield CH3: Protein is folded

2. 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 100-120 amino acids; spectra of larger proteins are too crowded

3. Useful nuclei such as 15N, 13C are rare Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance g/107 rad T-1s-1 MHz (2.3 T magnet) 1H 1/2 99.985 % 26.7519 100.000000 2H 1 0.015 4.1066 15.351 13C 1/2 1.108 6.7283 25.145 14N 1 99.63 1.9338 7.228 15N 1/2 0.37 -2.712 10.136783 17O 5/2 0.037 -3.6279 13.561 19F 1/2 100 25.181 94.094003 23Na 3/2 100 7.08013 26.466 31P 1/2 100 10.841 40.480737 113Cd 1/2 12.26 -5.9550 22.193173

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

5. Requirements for heteronuclear NMR: isotope 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 (15NH4Cl, 13C-glucose) or in labelled rich media. 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 CH3

6. Requirements for heteronuclear NMR: isotope labeling Deuterium labeling For large proteins deuterium labeling provides simplified spectra for the remaining 1H nuclei and has useful effects on relaxation properties of attached or adjacent atoms (fast relaxation=broadNMR lines) 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)

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

8. 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  Check folding Protein folding can be checked by 1H NMR and 1H-15N HSQC spectra.

9. 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 15N nitrogen source or/and 13C carbon source

10. 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 can beglucose (the best asgiveshighestyiels), glycerol, acetate, succinate, methanol, Etc. In case of13C labeling the concentrationofcarbon source can bereducedwithrespecttounlabelled culture, to reduce costs!!! Checksmustbeperformedbeforelabelling! Nitrogen source can be NH4Cl or (NH4) 2SO4 In case of15N labeling the concentrationofnitrogen source can bereducedwithrespecttounlabeled culture, to reduce costs!!! Checksmustbeperformedbeforelabeling!

11. 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. Saltsare NaCl/KCl, MgSO4, CaCl2 Buffer usuallyisphosphate, pH 7.5 Trace elementsisconstitutedbya mixturesof metal ions, like Co2+, Cu2+, Zn2+, Mn2+, Fe2+ Vitaminsare thiamine, biotin, folic acid, niacinamide, pantothenic acid, pyridoxal, riboflavin

12. 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.

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

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

15. 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

16. Strategies to improve protein expression In practice: Cells are grownin rich unlabeled medium. When OD600 = 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.

17. The need of deuteration Whyisnecessarytoenrich the proteinwith2H? Deuterationreduces the relaxationratesofNMR-active nuclei,in particular13C Itimproves the resolution and sensitivityof NMR experiments

18. 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) 13C/15N labeling  for c  up to 18 ns (35 KDa) 13C/15N labeling and fractional deuteration  for c above 18 ns 13C/15N labeling – selective protonation and background deuteration It depends from the type of NMR experiments

19. The problem to express a deuterated protein Incorporation of 2H reduces growth rate of organisms (up to 50%) and decreases protein production Deuteriumlabelingrequiresdifferentconditions withrespectto13C and 15N enrichment and couldrequirebacteriaadaptation

20. Fractional deuteration Randomfractionaldeuteration can beobtained up to a levelof 70-75%, in a media with 85% D2O with protonatedglucose, withoutbacteriaadaptation Expressing culture labeled >20 h Preinduction culture labeled 2-6 hours OD600=0.3-1.2 O/N culture unlabeled As for 13C-15N labelling all the conditions (strain, glucose conc. time of induction, etc.) must be optimized for each protein!!

21. Deuterium incorporation Fractional deuteration of recombinant proteins determined using mass spectroscopy. ( ) deuteration with [2H]2O only. ( ) deuteration with [2H]2O and perdeuterated glucose. O’Connell et al. Anal.Biochem. 1998, 265, 351-355

22. Perdeuteration Perdeuterationrequire a gradualadaptationof bacteriatoincreasingconcentrationof D2O. Bacterialstrainsmustbeaccuratelyselected in order tochoosethatwhichbetteracclimatesto D2O media. Foreach strain one or more colonymustbeselected whichbettersurvives in high levelof D2O concetration

23. A protocol for bacteria adaptation to deuterated medium 40% D2O 60% D2O 80% D2O 99 % D2O O/N Inoculum in unlabelled medium Massive culture 99% D2O Glycerol stock 40% D2O Glycerol stock 60% D2O Glycerol stock 80% D2O Glycerol stock 99% D2O

24. Is it possible to avoid the adaptation phase? Wüthrichlabhasexperimented a culture minimal medium supplementedwithdeuteratedalgalhydrolysatewhichallowsusto eliminate cellspre-conditioning Composition of the Celtone-supplemented media Basic minimal medium 800 ml H2O or D2O 100 ml M9 solution 2 ml 1M MgSO4 1 g NH4Cl 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 Wüthrich K. et al J.Biomol.NMR 2004,29,

25. Is it possible to avoid the adaptation phase? SOME RESULTS Medium composition Deuteration Advantage/disadvantages Minimal medium on 60-92% no N-H/N-D exchange problems Glucose-d + Celtone-d intermediate deuteration can be achieved in H2O Minimal medium on 95-97% high deuteration Glucose-d + Celtone-d in D2O Wüthrich K. et al J.Biomol.NMR 2004,29,

26. Backbone HN Side-chians

27. 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)

28. Specific labelling: the main problem The most important problem encountered is the metabolic conversion of the labeledaminoacids which might occur during anabolism and/or catabolism. How to prevent this? 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 13C15N Lys can be performed in unlabeled media with high level of 13C15N Lys to prevent lysine biosinthesis from aspartate conversion. However, if complete control over the incorporation of amino acids is required, then cell-free methods must be used.

29. Specific labeling for assignment of 13C and 1H methyl from Ile, Leu, Val 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

30. SAIL - Stereo-Array Isotope Labelling The basic strategy of the SAIL approach is to prepare amino acids with the following features: Stereo-selective replacement of one 1H in methylene groups by 2H. Replacement of two 1H in each methyl group by 2H. Stereo-selective modification of the prochiral methyl groups of Leu and Val such that one methyl is 12C(2H)3 and the other is 13C1H(2H)2. Labelling of six-membered aromatic rings by alternating 12C-2H and 13C-1H moieties The 20 protein-component SAIL amino acids are prepared based on these design concepts by chemical and enzymatic syntheses.

31. 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.

32. 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 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

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

34. 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 beachievedusinganauxotrophic strain defective in shikimate production

35. An example of Site-specific labelling To obtain CH3 in perdeuterated protein sample: α-Ketoacid Precursors for Biosynthetic Labeling of Methyl Sites [1H,13C]-labeled pyruvate as the main carbon source in D2O-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.

36. A bacterial protein expression system with 13C,1H pyruvate as the sole carbon source in D2O media

37. Unfortunately, because the protons of the methyl group of pyruvate exchange with solvent, proteins are produced with all four of the possible methyl isotopomers (13CH3, 13CH2D, 13CHD2, and 13CD3).

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

39. The protein is produced by expression from bacteria which are grown on minimal medium in D2O using 13C,2H-glucose as the main carbon source and 15NH4Cl 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.                                                                                               

40. 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 www.neb.com/neb/inteins.html) 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).

41. SEGMENTAL LABELLING

42. BUONO STUDIO!