A perspective on 13 C observe experiments: useful for paramagnetics, but how about diamagnetics?

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1 A perspective on observe experiments: useful for paramagnetics, but how about diamagnetics? Isabella. Felli ERM, University of Florence, Italy

2 direct detection - Paramagnetic R 1I, R 2 I γ Ι2 γ S 2 (1/r I6 ) f(τ e ) r H r Paramagnetic center 1 H γ Η2 /γ 2 (r /r H ) 6 detectable also at distances where 1 H broadened beyond detection! Bertini I., Luchinat., Parigi G., Solution NMR of paramagnetic molecules, Elsevier, 2001

3 direct detection - Paramagnetic R 1I, R 2 I γ Ι2 γ S 2 (1/r I6 ) f(τ e ) r H Paramagnetic center 1 H r γ Η2 /γ 2 (r /r H ) 6 1 H and paramagnetic relaxation rate enhancements provide complementary distance constraints! Bertini I., Luchinat., Parigi G., Solution NMR of paramagnetic molecules, Elsevier, 2001

4 direct detection u(ii)zn(ii) SOD Fast relaxation in paramagnetic proteins R 1I, R 2 I γ Ι2 γ S 2 (1/r I6 ) f(τ e ) 1 H 11 Å blind sphere!!! only ligands!!! u Zn 1 H and paramagnetic relaxation rate enhancements as structural constraints Bermel W., Bertini I., Felli I.., Kümmerle R., Pierattelli R., J. Am. hem. Soc., 2003, 125, Bertini I., Felli I.., Luchinat., Parigi G., Pierattelli R., hem. Bio. hem, 2007, 8, H

5 direct detection - Paramagnetic opper Type II u(ii) alcium substitution with lanthanides Iron Heme, non-heme H32 H83..a recent review: Bertini, I.; Luchinat,.; Parigi, G.; Pierattelli, R. Dalton Trans. 2008, Y75

6 direct detection Paramagnetic 1 H e - Fast relaxation in paramagnetic proteins R 1I, R 2 I γ Ι2 γ S 2 (1/r I6 ) f(τ e ) 1 H e - Bermel W., Bertini I., Felli I.., Kümmerle R., Pierattelli R., J. Am. hem. Soc., 2003, 125, Bertini I., Felli I.., Luchinat., Parigi G., Pierattelli R., hem. Bio. hem, 2007, 8, Madl T., Felli I.., Bertini I., Sattler M., J. Am. hem. Soc., 2010, 2,

7 direct detection Paramagnetic tags BAO spectra of [U-2H,15N,]-U2AF65-N155 in complex with U9 RNA. The nitroxide spin label is covalently attached to ys155. Madl T., Felli I.., Bertini I., Sattler M., J. Am. hem. Soc., 2010, 2,

8 direct detection general tool? Heteronuclear relaxation Heteronuclear chemical shift dispersion Why not as a general tool also for biomolecular NMR applications?

9 Increase sensitivity at ERM :1 (TXI) :1 (TXO*) :1 (TI*) :1 (cryo-txo*) :1 (cryo-txo*) *Thanks to R. Kümmerle, W. Bermel, M. Fey, K. Kessler, T. Gianotti, O. Schett!

10 direct detection - how long? start, detect (1 mm ubiquitin) AO ns=1 ON ns=1

11 The suite of observe experiments Some reviews Bermel W., Bertini I., Felli I.., Piccioli M., Pierattelli R., Progress in NMR spectroscopy, 2006, 48, Bermel W., Felli I.., Kümmerle R., Pierattelli R., oncepts in magnetic resonance, 2008, 32A, hapters from the book: NMR of Biomolecules: Towards Mechanistic Systems Biology, Bertini I, McGreevy K, Parigi G (Eds.), Wiley, 2012 hapter 7 - Felli, I..; Pierattelli, R and Tompa P.; Intrinsically disordered proteins hapter 25 - Felli, I.. and Pierattelli, R; direct detection NMR Several variants that differ in: homonuclear decoupling start, 1 H start, 1 H flip Transfer types

12 detection homonuclear decoupling Solution and solid state NMR J α α ali aro J α J αβ

13 direct detection IPAP & S 3 E SUM IPAP DIFF DIFF SUM 90 IPAP S 3 E HN - Nielsen N.., Thøgersen H., Sørensen O.W., J. Am. hem. Soc., 1995, 117, 165-6, Ottiger M., Delaglio F., Bax A., J. Magn. Reson., 1998, 1, , Andersson P., Weigelt J., Otting G., J. Biomol. NMR, 1998, 12, , Meissner A., Duus J.O., Sørensen O.W., J. Magn. Reson., 1997, 128, solid-state - Duma L., Hediger S., Lesage A., Emsley L., J. Magn. Reson, 2003, 164, , Duma L., Hediger S., Brutscher B., Böckmann A., Emsley L., J. Am. hem. Soc., 2003, 125, , Verel R., Manolikas T., Siemer A.B., Meier B.H., J. Magn. Reson. 2007, 184, solution - Hu K., Eletsky A. Pervushin K., J. Biomol. NMR, 2003, 26, 69, Bertini I., Felli I.., Kümmerle R., Luchinat., Pierattelli R., J. Biomol. NMR, 2004, 30, , Bermel W., Bertini I., Duma L., Felli I.., Emsley L., Pierattelli R., Vasos P.R., Angew. hem., 2005, 44, , Bermel W., Bertini I., Felli I.., Matzapetakis M., Pierattelli R., Thiel E., Turano P., J. Magn. Reson., 2007, 188, , Bermel W., Felli I.., Kümmerle R., Pierattelli R., oncepts in magnetic resonance, 2008, 32A,

14 How about α? J α α J J αβ α α J J αβ α IPAP (J α ) IPAP (J α )& IPAP (J αβ ) S 3 E (J α )& S 3 E (J αβ ) IPAP (J α ) DIPAP (J α, J αβ ) DS 3 E (J α, J αβ ) Duma L., Hediger S., Lesage A., Emsley L., J. Magn. Reson, 2003, 164, Bermel W., Bertini I., Duma L., Felli I.., Emsley L., Pierattelli R., Vasos P.R., Angew. hem., 2005, 44, Bermel W., Bertini I., Felli I.., Matzapetakis M., Pierattelli R., Thiel E., Turano P., J. Magn. Reson., 2007, 188,

15 heteronuclear decoupling IPAP ( α ) 15 N decoupling IPAP ( α ) & SE-IPAP ( N) Nielsen N.., Thøgersen H., Sørensen O.W., J. Am. hem. Soc., 1995, 117, Kern T., Shanda P., Brutscher B. J.Magn. Reson., 2008, 190, Meissner A., Duus J.O., Sørensen O.W., J. Magn. Reson., 1997, 128, Bermel W., Bertini I., sizmok V., Felli I.., Pierattelli R., Tompa P., Ottiger M., Delaglio F., Bax A., J. Magn. Reson., 1998, 1, J. Magn. Reson., 2009, 198, Andersson P., Weigelt J., Otting G., J. Biomol. NMR, 1998, 12, Duma L., Hediger S., Lesage A., Emsley L., J. Magn. Reson, 2003, 164, Duma L., Hediger S., Brutscher B., Böckmann A., Emsley L., J. Am. hem. Soc., 2003, 125, Bertini I., Felli I.., Kümmerle R., Luchinat., Pierattelli R., J. Biomol. NMR, 2004, 30, Bermel W., Bertini I., Duma L., Felli I.., Emsley L., Pierattelli R., Vasos P.R., Angew. hem., 2005, 44,

16 detection which variant? exclusively heteronuclear experiments 15 N 15 N / 15 N start? 1 H start? 1 H flip?

17 detection which variant? 1 H DΔν( ) DΔν( 1 H) 15 N 15 N / 15 N start? 1 H start? 1 H flip?

18 detection which variant? 1 H Dδ( 1 H) Dδ( ) 15 N 15 N / 15 N start? 1 H start? 1 H flip?

19 direct detection - unfolded systems/idps When possible, 1 H polarization can still be used to increase sensitivity 15 N 15 N / 15 N Exclusively heteronuclear experiments! Serber Z., Richter., Dötsch V., hembiohem., 2001, 2, , Pervishin K., Eletsky A., J. Biomol. NMR, 2003, 25, Jordan J.B., Kovacs H., Wang Y., Mobli M., Luo R., Anklin., Hoch J.., Kriwacki R.W., J. Am. hem. Soc., 2006, 128, Fiala R., Sklenar V., J. Biomol. NMR, 2007, 39, , Farès., Amata I., arlomagno T., J. Am. hem. Soc., 2007, 129, Hu K., Vögeli B., lore G. M., J. Am. hem. Soc., 2007, 129 (2007) Bermel W., Bertini I., sizmok V., Felli I.., Pierattelli R., J. Magn. Reson., 2009, 198,

20 detection which variant? 1 H Dδ( 1 H) Dδ( ) 15 N 15 N / 15 N start? 1 H start? 1 H flip?

21 direct detection speeding up Longitudinal relaxation enhancement 1 H-start, 1 H-flip Diercks, T.; Daniels, M.; Kaptein, R. J.Biomol.NMR 2005, 33, Deschamps, M.; ampbell, I. D. J.Magn Reson. 2006, 178, Schanda, P.; Brutscher, B. J.Am.hem.Soc. 2005, 127, Müller, L. J.Biomol.NMR 2008, 42, Bermel, W., Bertini I., Felli I.., Pierattelli, R., J. Am. hem. Soc., 2009, 1,

22 direct detection speeding up -start 1 H α -start 1 H α -flip (Hflip)ON-IPAP Reducing d1 from 1.2s to 200m Bermel, W., Bertini I., Felli I.., Pierattelli, R., J. Am. hem. Soc., 2009, 1,

23 direct detection speeding up Reduction in datapoints acquired in indirect dimensions 3 days ½ day (Hflip)AO-IPAP ~2 min (Hflip)ANO -IPAP Bermel, W., Bertini I., Felli I.., Pierattelli, R., J. Am. hem. Soc., 2009, 1,

24 direct detection applications 15 N 15 N / 15 N Paramagentic Very large Intrinsically disordered Exchange

25 direct detection IDPs! u(i)zn(ii)sod 153 AA Well folded Synuclein 140 AA IDP

26 carbonyl direct detection IDPs H N i-n i i-1 -N i Zhang, H., Neal, S., Wishart, D.S., J. Biomol. NMR 2003, 25, Schwarzinger S., Kroon G.J., Foss T.R., hung J., Wright P.E., Dyson H.J., J. Am. hem. Soc. 2001, 123,

27 Securin Intrinsically disordered protein Interphase Prophase Prometaphase Metaphase Anaphase Telophase ytokinesis Securin inhibitor of separase Metaphase Securin Intrinsically disordered protein (IDP!) 202 AA (>10% PROs) Anaphase

28 Securin - Intrinsically disordered protein GLY (N) 9 corr obs GLY (N) 11 corr obs PRO (N) 22 corr obs HSQ: % of the expected 60% of the whole protein ON: % of the expected 82% of the whole protein sizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. hem. Soc., 2009, 0,

29 (H)BAON-IPAP of securin (IDP) ALA PRO GLY SER/THR PRO PRO X-Pro orrelations observed: α i, i,n i+1, β i, i,n i MHz cryotxo, 21h 0.7 mm Bermel, Bertini, sizmok, Felli, Pierattelli, Tompa, J Magn Reson, 2009 sizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. hem. Soc., 2009, 0,

30 direct detection Unfolded/IDPs Set of experiments for sequence specific assignment 15 N 15 N / 15 N 1 H / 15 N ON-IPAP (H)BAON-IPAP (H)BANO-IPAP (H)NANO-IPAP OON-MOA-IPAP HSQ HNO BAONH BANH HN(A)NNH 97%, 99% α and β, 97% H N, and 98% N > 97%

31 Human securin - other NMR observables D 150 -F 159 E 1 -S 127 W 174 -L 178 sizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. hem. Soc., 2009, 0,

32 Heterogeneous behaviour of ox17 ox17 SS Oxidized (2 S-S bonds) ox17 SH Reduced (no S-S bonds) Helix I Helix II Arnesano, F.; Balatri, E.; Banci, L.; Bertini, I.; Winge, D. R. Structure 2005,, Bertini I., Gonnelli L., Felli I.., V. Kumar, Pierattelli, R., Angew.hem.Int.Ed. 2011, 50, Bertini I., Gonnelli L., Felli I.., V. Kumar, Pierattelli, R., hembiohem, e-pub

33 haracterization of IDPs ox17 SH hapters from the book: NMR of Biomolecules: Towards Mechanistic Systems Biology, Bertini I, McGreevy K, Parigi G (Eds.), Wiley, 2012 hapter 7 - Felli, I..; Pierattelli, R and Tompa P.; Intrinsically disordered proteins hapter 25 - Felli, I.. and Pierattelli, R; direct detection NMR

34 direct detection exchange rates H 2 O Increasing mixing time Bertini I., Gonnelli L., Felli I.., V. Kumar, Pierattelli, R., hembiohem, e-pub

35 direct detection ox17y SH ox17y SS Oxidized form Overall picture No exchange ox17y SH Reduced form H/HN NOE hemical shifts - SI

36 direct detection in-cell heteronuclear chemical shift dispersion...how about in-cell applications? an direct detection be of some use?

37 direct detection in-cell Folded (Atx1) Unfolded (α-synuclein) purified in-cell purified in-cell HN SOFAST (cryotxi) ns=16 d 1 =300m td1=128 ns=16 d 1 =300ms td1=256 Bertini I., Gonnelli L., Felli I.., V. Kumar, Pierattelli, R., Angew.hem.Int.Ed. 2011, 50,

38 direct detection in-cell purified, reduced ox17y SH in-cell ox17y purified, oxidized ox17y SS HN SOFAST (cryotxi), ns=16, d 1 =300m, td1=128 The protein is reduced in-cell! Banci L., Bertini I., efaro., enacchi L., iofi-baffoni, S., Felli I.., Gallo A., Gonnelli L., Luchinat, E., Sideris D., Tokatlidis K., PNAS 2010, 107, Bertini I., Gonnelli L., Felli I.., V. Kumar, Pierattelli, R., Angew.hem.Int.Ed. 2011, 50,

39 direct detection in-cell in-cell purified in-cell ns=16, d 1 =300ms, td1=256 Bertini I., Gonnelli L., Felli I.., V. Kumar, Pierattelli, R., Angew.hem.Int.Ed. 2011, 50, ns=8, d 1 =900ms, td1=128

40 Summary and conclusions omplementary general tool also in biomolecular NMR In several cases it anables us to recover lost information! Paramagnetic proteins Unfolded proteins/idps Nice probes at ERM! Access to research infrastructures EASTNMR BioNMR

41 Acknowledgments Ivano Bertini Roberta Pierattelli Wolfgang Bermel Rainer Kümmerle ERM!