Antoine Loquet
Transcription
Antoine Loquet
Structure determination of fully labeled proteins by solid-state NMR Antoine Loquet IBCP-CNRS Lyon, France [email protected] . Distance restraints on fully 13C, 15N labeled proteins . Handle highly ambiguous solid-state NMR data . Application to high-resolution structure calculation of the Crh protein . Additional restraints: JCHHC experiment . Carbon-carbon correlation for 3D structure determination: TSAR [email protected] . Distance restraints on fully 13C, 15N labeled proteins . Handle highly ambiguous solid-state NMR data . Application to high-resolution structure calculation of the Crh protein . Additional restraints: JCHHC experiment . Carbon-carbon correlation for 3D structure determination: TSAR [email protected] Internuclear distance measurements ? 1 H ...1H large dipolar coupling: broad lines in proton spin-diffusion exp. Biochemical tricks: spin dilution (Reif / Zilm / Rienstra) i.e. high-resolution structure of GB1 with back-exchanged1 H sample Zhou et al., Angew. Chem. Int. Ed. 2008 [email protected] Internuclear distance measurements ? 1 H ...1H large dipolar coupling: broad lines in proton spin-diffusion exp. Biochemical tricks: spin dilution (Reif / Zilm / Rienstra) i.e. high-resolution structure of GB1 with back-exchange 1H sample Zhou et al., Angew. Chem. Int. Ed. 2008 13 C ... C dipolar truncation Hohwy et al., J. Chem. Phys 2002 Grommek et al., Chem. Phys. Lett. 2006 Number of contacts 13 Loquet et al., J. Am. Chem. Soc 2008 300 150 0 1.5 [email protected] 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Carbon-carbon distance / Å sequential (|i-j|=1) long-range (|i-j|>4) intraresidue medium-range (1<|i-j|<4) 6.0 6.5 7.0 Internuclear distance measurements ? 1 H ...1H large dipolar coupling: broad lines in proton spin-diffusion exp. Biochemical tricks: spin dilution (Reif / Zilm / Rienstra) i.e. high-resolution structure of GB1 with back-exchange 1H sample Zhou et al., Angew. Chem. Int. Ed. 2008 13 C ... C dipolar truncation Hohwy et al., J. Chem. Phys 2002 Grommek et al., Chem. Phys. Lett. 2006 few kHz coupling Number of contacts 13 Loquet et al., J. Am. Chem. Soc 2008 300 150 0 1.5 2.0 2.5 -> 1-bond / 2-bonds dominate [email protected] 3.0 3.5 4.0 4.5 5.0 5.5 Carbon-carbon distance / Å sequential (|i-j|=1) long-range (|i-j|>4) intraresidue medium-range (1<|i-j|<4) 6.0 6.5 7.0 Pulse sequences for fully labeled system . Spin diffusion, DARR, RAD Franks et al., PNAS 2007 Manolikas et al., J. Am. Chem. Soc 2008 . Proton mediated, rare spin detected Lange et al., Angew. Chem. Int. Ed. 2008 / Korukottu et al., PloS ONE 2008 Loquet et al., J. Am. Chem. Soc 2008-a / Loquet et al., J. Am. Chem. Soc 2008-b H C H C H C H C H . Third Spin Assisted Recoupling De Paëpe et al., accepted [email protected] C C Proton mediated, rare spin detected: N/CHHC 10 Lange et al., J. Am. Chem. Soc 2002 Lange et al., J. Am. Chem. Soc 2003 ! 2 1H 13C CP spinal64 CP CP t1 CP 15N mixing time ! 2 ! 2 1H ! 2 CP CP 13C spinal64 t1 CP 20 CP spinal64 CP t2 ! 2 mixing time CP spinal64 CP CP t2 Carbon 13 chemical shift / ppm ! 2 30 40 50 60 70 60 40 20 Carbon 13 chemical shift / ppm CHHC, mixing time: 200µs at 500 MHz, 4 mm rotor fully labeled Crh protein [email protected] Proton mediated, rare spin detected: N/CHHC 10 1. Intensity <-> distance ? 2. Enough information for 3D structure calculations ? 3. Deal with high-level of ambiguity Carbon 13 chemical shift / ppm 20 30 40 50 60 70 60 40 20 Carbon 13 chemical shift / ppm CHHC, mixing time: 200µs at 500 MHz, 4 mm rotor fully labeled Crh protein [email protected] 12 0.8 12-13 43-44 62-63 65-66 26-27 47-48 48-49 51-52 77-78 H(i) Cα N C Cα 9 Signal intensity / a. u. Peak intensity / a.u. 1. Build-up magnetization curves Hα(i-1) O 6 Cα N 3 0 Hα(i-1) H(i) C Cα O 0 20 40 60 80 0.6 0.2 0 100 Mixing time / µs <3Å 3-4 Å >4Å 0.4 0 100 200 Proton-proton mixing time / µs 300 360 N H 2.18 Å H Leu10 300 γ χ2 240 HN 180 120 possible for simple case: 3.88 Å Leu74 60 0 Peak intensity / a.u. 0.8 0.6 0.4 0.2 0 0 intraresidue sequential one H attached Signal intensity / a. u. 1 60 120 180 χ1 240 300 360 Hγ 0 50 100 1H-1H mixing time / µs 150 L10 (2.18 Å) L53 (4.60 Å) L74 (3.88 Å) 6 an unique upper distance, 4 2 no distance classes for calculations 0 0 [email protected] 25 50 75 Mixing time / µs 100 Gardiennet, Loquet, Etzkorn, Heise, Baldus, Böckmann J. Biomol. NMR 2008 40(4), 239-250 2. Assignment using back-calculated spectra Carbon 13 chemical shift / ppm 20 40 60 80 54CB-55CB 47CD1-19CA 64CD1-35CA 44CB-77CB 64CD1-41CD 51CE-14CA 42CG2-40CE 64CD1-62CG2 51CE-19CB 64CD1-62CA 54CB-13CA 1CE-2CA 26CB-22CA 47CG1-48CG 33CG1-65CA 65CB-31CA 26CB-27CB 55CG1-54CA 61CG1-59CA 55CG2-54CB 53CG-52CB 41CG-35CB 74CG-63CB 37CD-59CA 17CB-18CD 40CD-39CA 45CD-44CA 64CG1-62CB 23CB-47CD1 68CB-31CB 45CD-31CA 60CB-59CA 60CG-59CA 66CG-31CB 47CB-50CB 66CB-65CA 61CB-62CA 22CB-50CB 75CG-78CB 36CB-62CB 42CB-44CA 36CB-35CB 41CE-64CB 55CB-54CA 47CB-20CB 47CB-46CB 29CB-73CA 45CE-29CB 29CB-30CA 22CB-44CB 29CB-30CB 77CB-63CB 45CE-27CB 35CB-53CG 32CB-31CA 37CE-59CA 13CA-55CB 64CB-65CA 32CB-31CB 14CB-51CE 63CB-61CB 13CA-14CB 63CB-35CA 58CA-57CB 63CB-78CB 63CB-62CB 35CB-42CG1 63CB-62CA 67CA-68CB 13CA-55CA 63CB-77CA 63CB-35CB 54CA-55CA 35CB-14CD1 49CA-42CB 49CA-51CA 69CA-67CA 60CA-62CA 65CA-66CG 44CA-23CG2 54CA-37CE 18CD-14CA 46CA-47CA 54CA-37CB 20CA-47CD1 34CA-35CB 56CA-59CA 40CA-39CA 69CA-73CA 69CA-68CB 14CA-18CA 56CA-13CA 46CA-47CG1 32CA-65CA 29CA-30CB 48CA-47CG1 25CA-23CA 74CA-63CB 45CA-44CA 48CA-51CE 25CA-22CA 62CA-64CG1 85CA-79CB 42CA-49CA 64CA-62CB 12CA-54CA 31CA-33CG2 52CA-49CA 68CA-31CB 30CA-45CD 22CA-23CA 31CA-33CA 31CA-67CA 55CA-59CA 81CA-79CB 18CA-14CG 31CA-32CA 31CB-30CB 62CA-36CB 31CA-30CA 59CA-58CA 23CA-45CD 47CA-46CB 30CA-68CG 31CB-33CA 31CB-33CB 59CB-58CA 30CB-31CA 62CB-61CB 47CA-51CA 31CA-29CA 18CA-22CB 59CB-55CA 31CA-45CE 30CA-29CA 80CA-77CB 62CB-61CA 52CB-40CE 81CA-77CA 18CA-14CB 80 40 60 20 Carbon 13 chemical shift / ppm DARR (mixing: 200ms) 4 mm rotor 500 MHz [email protected] Simulated spectra to assign restraints in CHHC, NHHC and DARR: 0.25 ppm tolerance 1 possible assignment cutoff 5Å for H...H correlation cutoff 7Å for C...C correlation 2. Assignment using back-calculated spectra CC HH 2.5 Å 2.5 Å CC+HH 1.6 Å + TALOS 1.5 Å [email protected] 1.4 Å 1.3 Å Loquet, Bardiaux, Gardiennet, Blanchet, Baldus, Nilges, Malliavin, Böckmann J. Am. Chem. Soc. 2008 130(11) 3579-3589 < 1 % cross-peaks are assignable de novo structure determination ? deal with high-level of ambiguity 2 problems 1. each cross-peak is highly ambiguous in its CS assignment 2. each cross-peak could be intra or/and intermonomeric pdb entry: 1mu4 Juy et al., J. Mol. Biol. 2003 [email protected] Deal with ambiguity 1. Ambiguity treatment N -6 -1/6 D = (! d ) ADR (Ambiguous Distance Restraints) Nilges, J. Mol. Biol. 1995 a=1 -6 -6 -1/6 D = ( d intra +dinter ) 2. Asymmetric labeling Weiss, J. Magn. Reson. 1990 Arrowsmith et al., Biochemistry 1990 application on heterogenously labeled Crh protein Etzkorn et al., J. Am. Chem. Soc 2004 25 intermonomeric H...H restraints Dimeric topology of microcrystals Crh protein: A B AA and BB AB and BA 2 ambiguities per cross-peak [email protected] Nilges, Proteins 1993 N -6 -6 -1/6 D = (! d intra + !dinter ) a=1 with more than 10 ambiguities for CS assignment manual peak-picking on: CHHC (200µs) NHHC (100µs) N¹⁵ chemical shift / ppm Input data for ARIA calculation 100 a 120 140 70 20 Carbon 13 chemical shift / ppm Talos dihedral angle restraints no H bonds, homology model, etc... 50 40 30 20 Carbon 13 chemical shift / ppm treated as ADR, one distance class 1.8-5.0Å 20 intermonomeric NHHC distance restraints 60 b 30 40 50 60 70 70 60 50 40 30 Carbon 13 chemical shift / ppm Loquet et al., J. Am. Chem. Soc. 2008 [email protected] 20 Protocol ARIA 2.2: NCS C2 symmetry packing restraint slow cooling input 1st ARIA run best conformers template structures 2nd ARIA run unambiguous assignments xplor round ss-NMR structures [email protected] 1500 unambiguous ambiguous long-range count 1200 900 600 300 0 0 1 2 3 4 5 6 7 8 iteration 14 12 precision accuracy rmsd / Å 10 8 6 4 1st run 2 0 0 1 2 3 4 5 6 7 8 iteration rmsd / Å 8 precision accuracy 6 4 2nd run 2 0 0 1 2 3 4 5 iteration [email protected] 3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints Loquet, Bardiaux, Gardiennet, Blanchet, Baldus, Nilges, Malliavin, Böckmann J. Am. Chem. Soc. 2008 130(11) 3579-3589 ss-NMR structures [email protected] unambiguous distance restraints total sequential medium-range long-range 643 181 85 131 distance violations > 0.5 A none rmsd precision monomer, backbone dimer backbone 0.87 Å ± 0.12 Å 1.30 Å ± 0.20 Å accuracy monomer dimer 1.62 Å 2.89 Å 3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints Loquet, Bardiaux, Gardiennet, Blanchet, Baldus, Nilges, Malliavin, Böckmann J. Am. Chem. Soc. 2008 130(11) 3579-3589 new methods for distance restraints determination (also for large fully labeled system) ! 2 ! 2 1H CP spinal64 CP ! 2 mixing time CP spinal64 dipolar CHHC experiment (Lange et al., JACS 2002) 13C CP t1 CP CP t2 cross-polarization steps of short duration (100-200us) 1. selectivity of the CP transfer -> unexpected cross-peaks 2. 3 fold rotation of CH3 prevent efficient transfer -> lack of methyl-methyl contacts heteronuclear through-bond interaction J-CHHC experiment Carbon - carbons correlation which reflect proximities between their bonded H Methyl Proton Contacts Obtained Using Heteronuclear Through-Bond Transfers in Solid-State NMR Spectroscopy Loquet, Laage, Gardiennet, Elena, Emsley, Böckmann, Lesage J. Am. Chem. Soc. 2008 130(32) 10625-10632 200 180 160 140 120 100 80 60 40 20 0 Carbon 13 chemical shift / ppm Carbon 13 chemical shift / ppm 47Hδ-23Hγ 15 20 47Hδ-20Hβ 1 J-CHHC CHHC 64Hδ-62Hγ 51Hε-19Hβ 78Hβ-6Hγ 54Hβ-55Hγ 44Hβ-77Hδ 19Hβ-14Hδ 26Hβ-33Hγ 44Hβ-63Hδ 78Hβ-74Hγ 44Hβ-35Hδ 61Hγ-81Hγ 61Hγ-35Hδ 2Hγ-74Hγ 42Hγ-35Hδ 19Hβ-47Hδ 3 20Hβ-16Hβ 44Hβ-26Hβ 73Hβ-33Hγ 21Hδ-20Hβ 33Hγ-44Hβ 12Hγ-55Hγ 77Hδ-26Hβ 33Hγ-63Hδ 55Hγ-14Hδ 81Hγ-35Hδ 23Hγ-47Hγ 2 16Hβ-51Hε NHHC + CHHC precision: 1.5 Å accuracy: 1.8 Å 57Hγ-12Hγ 63Hδ-77Hδ 74Hδ-2Hγ 14Hδ-12Hγ 14Hδ-81Hγ 25 35Hδ-6Hγ 15 25 20 Carbon 13 chemical shift / ppm 30 1 * * 2 * * 3 ** * 35 30 25 20 15 10 35 30 25 20 15 10 35 30 25 20 15 10 13C chemical shift / ppm 13C chemical shift / ppm 13C chemical shift / ppm NHHC + CHHC + J-CHHC precision: 1.3 Å accuracy: 1.1 Å 1H CP TPPM C.W. TPPM highly reduce dipolar truncation 13C CP t1 C.W. t2 PAR Proton Assisted Recoupling and Protein Structure Determination De Paëpe, Lewandowski, Loquet, Böckmann, Griffin. JCP, accepted promote long distance contacts DARR 200ms PAR 15ms 20 Carbon 13 chemical shift / ppm Carbon 13 chemical shift / ppm 20 CHHC 200us 30 40 50 30 40 50 60 60 70 70 60 40 20 Carbon 13 chemical shift / ppm 60 40 20 Carbon 13 chemical shift / ppm PAHR 15ms PAHR 2ms Carbon 13 chemical shift / ppm 20 20kHz @ 900 MHz Crh protein 30 40 50 60 70 60 40 Carbon 13 chemical shift / ppm 20 De Paëpe, Lewandowski, Loquet, Böckmann, Griffin, JCP, accepted . High-resolution structure from solid-state NMR data deal with high-level of ambiguity CHHC / NHHC experiments for distance restraints . Additional restraints J-CHHC . 13C-13C correlation with efficient recoupling PAR Thanks IBCP (CNRS UMR 5086) Max-Planck-Institute for Biophysical Chemistry Massachussets Institute of Technology Anja Böckmann Marc Baldus Manuel Etzkorn Henrike Heise Adam Lange Bob Griffin Gael De Paëpe Josef Lewandowski Institut Pasteur ENS Lyon Michael Nilges Thérèse Malliavin Benjamin Bardiaux Anne Lesage Lyndon Emsley Ségolène Laage Carole Gardiennet LBRS
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