Dipole Recoupling at High Spinning Frequencies. and. High Magnetic Fields

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1 Dipole Recoupling at High Spinning Frequencies and High Magnetic Fields Stowe, Vermont January 2010 Francis Bitter Magnet Laboratory and Department of Chemistry Massachusetts Institute of Technology

2 Outline Recoupling at High Spinning Frequencies and Fields Dipole recoupling in the 20th century Recoupling sans decoupling Third spin assisted recoupling (TSAR) Applications to proteins ѡr/2π=65 khz Dynamic Nuclear Polarization (DNP) Instrumentation Biradical Polarizing Agents and DNP Mechanisms Uniform polarization of macroscopic samples

3 Dipole Recoupling Sequences DICSY SPC-5 SEDRA DRAMA RIL REDOR CROWN CMR7 C7 DRAWS USEME MELODRAMA TEDOR Rotor synchronized sequences developed for low fields 13 C- 13 C and 13 C- 15 N distance measurements

4 Radio Frequency Driven Recoupling (a.k.a. RFDR) Chemical shift correlation spectroscopy in rotating solids: Radio frequency-driven dipolar recoupling and longitudinal exchange A. E. Bennett, J. H. Ok, and R. G. Griffin Francis Bitter National Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts S. Vega Department of Chemical Physics, Weizmann Institute of Science, Rehovot Israel (Received 26 December 1991; accepted 23 March 1992) Homonuclear recoupling via a train of π pulses RFDR one of the first successful 2D recoupling techniques Bennett, Rienstra, Griffiths, Lansbury, and Griffin, J. Chem. Physics 108, 9463 (1998) Boender, Vega and degroot, J. Chem. Physics 112, 1096 (2000)

5 PI3-SH3 Band Selective (BASE)-RFDR PI3-SH3 protein fibrils - structural model Sequential assignments & long-range contacts alternating labeling with [2-13 C-glycerol] 20 ms low-bandwidth RFDR (12 khz 13 C rf) -./ # + #* ' '% '% $%%& '(! # π τ ), $%%&! " Cα-C0! Cα-Cα±1 BASE-RFDR yields Ca(i) -> Ca(i+1) and Cai -> Ca (i+2) x-peaks (~6 Å) --- only these are present in the spectrum

6 Structural Measurements in Peptides and Proteins Assignments Distances Torsion Angles Measure isotropic chemical shifts and CSA s Measure magnitudes of dipolar couplings Measure relative orientations of dipolar tensors Structural parameters are measured via reintroduction of the 13 C- 15 N and 13 C- 13 C dipolar couplings into MAS spectra

7 13 C=O- 13 C=O Distance Measurements DQ-DRAWS Experimental requirements: ω r /2π = 20 khz ω 1 /2π =100 khz for SPC5 ω 1 /2π =140 khz for C7, POSTC7 ω 1 /2π =170 khz for DRAWS N=8.5! ѡ1( 13 C)=8.5 x ѡr and ѡ1( 1 H)= 3 x ѡ1( 13 C) 1 H decoupling > x3 13 C field, i.e times the MAS frequency ~ khz!

8 TSAR DARR versus spinning frequency High fields requires high spinning frequencies ω r /2π 10 khz ω r /2π 20 khz Cross peaks are missing or attenuated at ω r /2π = 20 khz Lewandowski et al (in preparation)

9 TSAR Methods yielding long distance restraints 13 C- 13 C correlation spectra of [U- 13 C, 15 N]-Crh protein DARR / RAD 20 mg, 21 hours Takegoshi et al. CPL (2001) 344. Morcombe et al. JACS (2004) 126. CHHC 20 mg, 46 hours Lange et al., JACS (2002) 124. Loquet et al. JACS (2008) C- 13 C PAR 6 mg, 21.4 hours De Paëpe et al. (2008) Lewandowski et al. (2008) DARR/RAD and CHHC spectra courtesy of Carole Gardiennet

10 Dipole Recoupling Sequences DICSY SPC-5 SEDRA DRAMA HIGH ѡr/2π and ѡ0/2π RIL CROWN CMR7 C7 REDOR USEME MELODRAMA TEDOR Most of the rotor synchronized sequences developed for low fields < 500 MHz do not function well at high ω r /2π (and high B 0 )

11 Outline Recoupling at High Spinning Frequencies and Fields Dipole recoupling in the 20th century Recoupling sans decoupling Third spin assisted recoupling (TSAR) Applications to proteins ѡr/2π=65 khz Dynamic Nuclear Polarization (DNP) Instrumentation Biradical Polarizing Agents and DNP Mechanisms Uniform polarization of macroscopic samples

12 New Solution: DQ-CMRR Recoupling sans 1 H decoupling Properties/advantages: Single channel irradiation Attenuation of r.f. sample heating Efficient at high B 0, high ω r /2π 13 C chemical shift (ppm) 13 C- 13 C recoupling without 1 H decoupling, 20 khz MAS CM 5 RR* on [U- 13 C, 15 N]-Crh MHz, 15 hours Efficient relayed transfer mechanism 13 C 13 C chemical shift (ppm) *De Paëpe et al., JACS (2006) De Paëpe et al., JCP (2008)

13 13 C- 13 C 2D spectra sans 1 H Decoupling PI3-SH3 fibrils CM 4 RR at 900 MHz

14 PI3-SH3 Linewidths in U- 13 C, 15 N and 2-13 C*,U- 15 N labeled samples MAS NMR on PI3-SH3 fibril samples -- linewidths ~0.5 ppm expression of isotopically labeled proteins manipulation of labeling scheme, by using different sources of 13 C and/ or 15 N isotopes Example: alternating labeling using [1,3-13 C]- or [2-13 C]-glycerol Labeling Pattern for Valine: [1,3-13 C] & [2-13 C]* Maly, Bayro et. al. submitted (2009)

15 PI3-SH3 PI3-SH3 protein fibrils- assignments Sequential assignments RFDR, 750 MHz, ωr/2π= khz 0.8 ms mixing 75/86 residues sequentially assigned 87% assigned! Excellent resolution ppm linewidths Maly, Bayro et. al. submitted (2009)

16 13 C- 13 C 2D spectra sans 1 H decoupling G B1 RFDR in the high frequency MAS/ high field regime Broadband ZQ 13 C- 13 C RFDR recoupling at 750 MHz without 1 H irradiation

17 Homonuclear Recoupling sans Decoupling RFDR 30 khz τ mix = 8 ms At high MAS rates, RFDR can be applies for extended mixing times without decoupling.

18 Outline Recoupling at High Spinning Frequencies and Fields Dipole recoupling in the 20th century Recoupling sans decoupling Third spin assisted recoupling (TSAR) Applications to proteins ѡr/2π=65 khz Dynamic Nuclear Polarization (DNP) Instrumentation Biradical Polarizing Agents and DNP Mechanisms Uniform polarization of macroscopic samples

19 Third Spin Assisted Recoupling Mechanism (TSAR) Pulse sequences Lewandowski, De Paëpe, Griffin JACS (2007) De Paëpe, Lewandowski, Loquet, Bockmann Griffin JCP (in press) (2008) Superficially similar to Hartmann-Hahn cross polarization RF fields satisfy different matching conditions

20 Proton Assisted Recoupling (PAR) [a.k.a. Third Spin Assisted Recoupling(TSAR)] PAR functions via second order cross terms -- not direct 13 C- 13 C terms Hint=ωN1N2(3N1zN2z-N1*N2) +ωn1h2n1zhz+ωhn22hzn2z Polarization transfer driven by... N 1 ± N 2 H z One bond and sequential cross peaks observed in 20 ms of mixing PDSD requires 4 sec of mixing for an equivalent spectrum! (Reif, et. al. 2000) Lewandowski, De Paepe and Griffin JACS 129, (2007); JACS 131, (2009)

21 Homonuclear TSAR Mechanism Second order average Hamiltonian theory ZQ flip-flop cross terms (2 x 3) lead to PAR transfer! Auto cross terms arise from 2 x 2 and 3 x 3 and attenuate PAR De Paëpe, Lewandowski, Loquet, Bockmann, Griffin JCP 129, (2008)

22 PAR Experiment SpinEv Optimization Map rotary resonance Hartmann-Hahn PAR p H =ω 1H /ω r ω r /2π=20 khz ω 0 /2π=750 MHz p C =ω 1C /ω r Appropriate choice of 13 C and 1 H rf leads to PAR recoupling!

23 Long Distance Constraints TSAR versus dipolar truncation Long distance transfer (~4.5 Å) in presence of directly bonded carbon: reduction of the dipolar truncation!

24 High field (750/900 MHz) Protein Structure Determination 33Cγ1/74Cδ1 6.8 Å Long distance transfer in uniformly labeled protein

25 de novo 3D Structure Determination Crh dimer X-ray Crh assignments + 2, 20 ms 13 C- 13 C PAR 15 ms PAIN-CP ARIA C- 13 C constraints (269 long range) N 13 C constraints (130 long range) TALOS XPLOR-NIH NMR RMSD = 0.58 Å

26 Outline Recoupling at High Spinning Frequencies and Fields Dipole recoupling in the 20th century Recoupling sans decoupling Third spin assisted recoupling (TSAR) Applications to proteins ѡr/2π=65 khz Dynamic Nuclear Polarization (DNP) Instrumentation Biradical Polarizing Agents and DNP Mechanisms Uniform polarization of macroscopic samples

27 TSAR High MAS frequency experiments: ѡr / 2π = 65 khz In collaboration with Jochem Struppe and Werner Maas (Bruker-Biospin) Expansion of aromatic region U- 13 C, 15 N GB1 1.3 mm rotor Low power decoupling khz Improved resolution Need for recoupling techniques

28 PAR Optimization ѡr/2π=65 khz 1.3 mm rotor Low power decoupling -- ѡ1/2π=16 khz Two regions that satisfy the matching conditions

29 Proton Assisted ѡr/2π=65 khz 13 C- 13 C PAR -- U( 13 C, 15 N)-GB1 Long distance transfer in uniformly labeled protein Resolved 13 C- 13 C J- couplings C=O to aliphatic Long distance contacts are observed in the aliphatic region SPINEV script for calculating transfer map M. Eddy, J. Struppe and W. Maas J. Phys. Chem. B, 113, (2009)