1H / Ultrafast MAS / Paramagnetic

Bernd Reif

Technische Universität München Helmholtz-Zentrum München

Biomolecular Solid-State NMR Winter School Stowe, VT January 07-12, 2018

1. Protons in Solid-State NMR

1) Ultrafast MAS / low power decoupling

2) Application of 1H,1H homonuclear decoupling schemes like FSLG/PMLG, DUMBO ...

3) Deuteration

4) Even faster MAS

Ultrafast MAS

Samoson, Tuherm, Gan, Solid State NMR 20 130 (2001) Ernst, Samoson, Meier, J. Magn. Res. 163, 332 (2003) Ernst, Meier, Tuherm, Samoson, Meier, J. Am. Chem. Soc. 126 4764 (2004) Samoson, Tuherm, Past, Reinhold, Anupold, Heinmaa, Top. Curr. Chem. 246 15 (2005) Laage, Sachleben, Steuernagel, Pierattelli, Pintacuda, Emsley, J. Magn. Res. 196 133 (2009)

1 H L

inew

idth

[Hz]

1H line widths of NH3, CH and CH3 in alanine, measured at 600 MHz

Proton Detection Heteronuclear Decoupling

MAS=60 kHz

Low-Power XiX Decoupling Schemes at Ultrafast MAS frequencies

Detken, Hardy, Ernst, Meier, Chem. Phys. Lett. 356 298 (2002) Ernst, Samoson, Meier, J. Magn. Reson. 203 332 (2003)

high-power XiX:

Gly

Cα

inte

nsity

Optimum for: τp = ca. 2.85 τr

low-power (XiX)45: MAS = 50 kHz τp = 76 µs ν1 = 13 kHz

high-power XiX: MAS = 50 kHz τp = 57 µs ν1 = 220 kHz

Low-Power Sequences for Hartmann-Hahn transfer n=0 Hartmann-Hahn match condition (ωI = ωS):

Second Order-CP (SOCP)

Lange, Scholz, Manolikas, Ernst, Meier, Chem. Phys. Lett. 468 100 (2009)

1H spin-lock efficiency as a function of the applied rf-field amplitude, indirectly detected by cross polarization to the Cα resonance of glycine ethyl ester (MAS = 65 kHz)

Implementation of Low-Power Building-Blocks in Multidimensional Solid-State NMR experiments

Vijayan, Demers, Biernat, Mandelkow, Becker, Lange ChemPhysChem 10, 2205 (2009)

Frequency Switched Lee-Goldburg (FSLG) and Phase Modulated Lee-Goldburg (PMLG) Experiments

for homonuclear 1H,1H Decoupling

To obtain decoupling, magnetization has to be rotated around the magic angle in spin space:

PMLG spectrum of a 15N labeled sample of a SH3 domain

15N Detection 1H Detection

Sensitivity Gain = x 9.0 @ 33 kHz MAS

€

S /N( ) 1H[ ]S /N( ) 15N[ ]

∝γ HγN

$

% &

'

( )

3 / 2

≈ 10( )3 / 2 = 30

€

S /N( )∝ nγ exc γDet3 B0

3t

JMR 151, 320-327 (2001); JMR 160, 78-83 (2003)

Protons in Solid-State NMR: Sensitivity and Resolution

u-[2H,15N]-Nac-Val-Leu-OH

Proton density in the deuterated α-spectrin SH3 domain

perdeuterated with 100% of labile protons back-exchanged

fully protonated

Chevelkov et al. J. Am. Chem. Soc. 125, 7788 (2003)

500 MHz, 10 kHz MAS

Proton density in the deuterated α-spectrin SH3 domain

perdeuterated with 10% of labile protons back-exchanged

Chevelkov et al. Angew. Chem. Int. Edt. 45 3878-3881 (2006)

Proton dilution in small organic molecules

Zheng, Fishbein, Griffin, Herzfeld, J. Am. Chem. Soc. 115, 6254 (1993)

1H

1% protonated Ala (= 99% deuterated) MAS = 11 kHz B0 = 360 MHz

1H

MREV-8 CRAMPS spectrum

McDermott, Creuzet, Kolbert, Griffin, J. Magn. Reson. 98, 408 (1992)

Deuteration in Solid-State NMR

Paulson, Morcombe, Gaponenko, Dancheck, Byrd & Zilm, Sensitive High Resolution Inverse Detection NMR Spectroscopy of Proteins in the Solid State. J Am Chem Soc 125: 15831-15836 (2003)

Morcombe, Gaponenko, Byrd & Zilm, C-13 CP MAS spectroscopy of deuterated proteins: CP dynamics, line shapes, and T-1 relaxation. J Am Chem Soc 127: 397-404 (2005).

Zhou & Rienstra, High-Performance Solvent Suppression for Proton-Detected Solid-State NMR. J Magn Reson 192: 167–172 (2008).

Zhou, Shah, Cormos, Mullen, Sandoz & Rienstra, Proton-detected solid-state NMR Spectroscopy of fully protonated proteins at 40 kHz magic-angle spinning. J Am Chem Soc 129: 11791-11801 (2007).

Schanda, Huber, Verel, Ernst & Meier Direct Detection of 3hJNC Hydrogen-Bond Scalar Couplings in Proteins by Solid-State NMR Spectroscopy. Angew Chem Int Edt 48: 9322-9325 (2009)

Schanda, Meier, Ernst Quantitative Analysis of Protein Backbone Dynamics in Microcrystalline Ubiquitin by Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 132: 15957–15967 (2010).

Solvent Suppression in the Solid-State

Chevelkov et al. JACS 125 7788 (2003)

a,b: zg c: x-filter d: x-filter + gradients

Solvent Suppression in the Solid-State

Solution 3: MISSISSIPPI Rienstra and co-worker JMR 192 167 (2008)

Solution 2: Zilm and co-worker JACS 125 15831 (2003)

High power RF irradiation

Imagine what happens to your sample (40 µL) when you apply 100 W for 30 ms ...

Using the water chemical shift as a thermometer

Sample Heating due to Friction (3.2 mm rotor)

"Static" Sample heating due to RF irradiation

Total time of irradiation d1 = 30 ms ωrf = 100 kHz, 100 mM NaCl recycle delay = 3s regular CP-MAS probe

a: no irradiation b: d2 = 2.7 s c: d2 = 1.7 s d: d2 = 1.0 s e: d2 = 0.03 s

Static/Steady-state sample heating due to RF irradiation (cw = 30 ms)

"Dynamic" Sample heating due to RF irradiation

Total time of irradiation d1 = 30 ms ωrf = 100 kHz, 100 mM NaCl recycle delay = 3s d5 = 1 ms (switching delay) regular CP-MAS probe

a: no irradiation b: t1 = 0 ms c: t1 = 10 ms d: t1 = 20 ms e: t1 = 30 ms

Dynamic sample heating in an indirect evolution period due to RF irradiation

d3 = 3s

What can you do with protons in the solid-state ?

Solution-State like Pulse Schemes applied to Crystalline Proteins

Increased reliability in the assignment of resonances in MAS solid-state NMR

HNCACB

Coherences are sufficiently long-lived in the solid-state to enable scalar transfers combined with 1H detection

Linser, Fink, Reif J. Magn. Reson. 193, 89 (2008) Linser, Fink, Reif, J. Biomol. NMR 47, 1 (2010)

More assignment experiments

Linser, R., Fink, U., and Reif, B. (2010). Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in the solid-state. J Biomol NMR 47, 1-6.

Linser, R. (2011). Side-chain to backbone correlations from solid-state NMR of perdeuterated proteins through combined excitation and long-range magnetization transfers. J Biomol NMR 51, 221-226.

Linser, R. (2012). Backbone assignment of perdeuterated proteins using long-range H/C-dipolar transfers. J Biomol NMR 52, 151-158.

Barbet-Massin, E., Pell, A.J., Jaudzems, K., Franks, W.T., Retel, J.S., Kotelovica, S., Akopjana, I., Tars, K., Emsley, L., Oschkinat, H., Lesage, A., and Pintacuda, G. (2013). Out-and-back C-13-C-13 scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast MAS. J Biomol NMR 56, 379-386.

Barbet-Massin, E., Pell, A.J., Retel, J.S., Andreas, L.B., Jaudzems, K., Franks, W.T., Nieuwkoop, A.J., Hiller, M., Hagman, V., Guerry, P., Bertarello, A., Knight, M.J., Felletti, M., Le Marchand, T., Kotelovica, S., Akopjana, I., Tars, K., Stoppini, M., Bellotti, V., Bolognesi, M., Ricagno, S., Chou, J.J., Griffin, R.G., Oschkinat, H., Lesage, A., Emsley, L., Herrmann, T., and Pintacuda, G. (2014). Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning. J Am Chem Soc 136, 12489-12497.

Chevelkov, V., Habenstein, B., Loquet, A., Giller, K., Becker, S., and Lange, A. (2014). Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins. J Magn Reson 242, 180-188.

HANAH (1H Natural Abundance in 2H Proteins)

Residual Protonation in perdeuterated proteins (> 97% 2H, 99% 13C -Glucose) 10% labeling of -CD2H

Residual Protonation in Perdeuterated Proteins

α-spectrin SH3

Methyl 1H,13C Correlations at 1H „Natural Abundance“

1H,13C double CP 1H,13C HMQC

Agarwal et al. JMR 194 16 (2008)

Increased sensitivity using specific precursors for amino acid biosynthesis

D

D

H

C

Goto, Gardner, Mueller, Willis, Kay, JBNMR 13, 369 (1999)

Agarwal, Diehl, Skrynnikov, Reif, JACS 128, 12620 (2006) Agarwal, Xue, Reif, Skrynnikov, JACS 130, 16611 (2008)

α-ketoisovalerate: Sparse labeling at Leu, Val sites (no measurable 13C-13C spin diffusion)

α-spectrin SH3

Detection of Exchangeable Hydroxyl Protons

α-spectrin SH3

W41ε

?

Exchangeable Hydroxyl Protons in α-spectrin SH3

13C detected 1H,13C-CP

HN

C´ Cα

∼ 1 ms

Exchangeable Hydroxyl Protons in α-spectrin SH3

Exchangeable Hydroxyl Protons in α-spectrin SH3

13C detected 1H,13C-CP

Agarwal et al. JACS 132 3187 (2010)

Exchange Properties of Hydroxyl Protons (α-SH3, 5°C)

Hydrogen Bonds in α-spectrin SH3: Y15(OH)-E22(COO-)

Hydrogen Bonds in α-spectrin SH3: Y15(OH)-E22(COO-)

Hydrogen Bonds in α-spectrin SH3: T24(OH)-E17(COO-)

0.94

0.53

€

DH , C = −µ0γ HγC!rH , C

3

€

Int C_O_H

Int H...O= C

τmix →∞( ) = DC _O _ H

DH ...O=C

= rH ...O=C

rC _O _ H

%

& '

(

) *

3

r(OH) = ca. 1.08-1.10 Å

Huber et al. Meier Chem Phys. Chem. 12, 915-918 (2011)

4D Solid-State NMR Experiment for Structure Determination

Linser et al. JACS 133, 5905-5912 (2011)

Use of time shared evolution periods in 3D/4D experiments

2D F3/F4 plane (red) of the 4D HN…NH

High Resolution Deuterium MAS solid-state NMR spectroscopy

1) Analysis of Side Chain and Backbone Dynamics

2) In Solution-State, 2H resonances are broad due to the J(0) contribution to 2H T2 CQ = ca. 55 kHz (methyl) and 165 kHz

€

R(D+) =180

e2qQ!

"

# $

%

& '

2

9J(0) +15J(ωD ) + 6J(2ωD )[ ]

J(ω) =19S2 τR1+ (ωτR )

2 + (1− 19S2)

τ f

1+ (ωτ f )2

In solution:

In solids

Deuterium Single Quantum (SQ) Correlations

Setup-System: 2H,13C,15N-NAc-Val-Leu-OH

Deuterium Double Quantum (DQ) Correlations

Setup-System: 2H,13C,15N-NAc-Val-Leu-OH

Making use of the spin-1 properties of 2H:

> Factor 2 increased resolution in the DQ experiment

τ = 3 µs (black), 9 µs (red/blue)

€

HQ =ωQ

33Iz Iz − I ⋅ I[ ]

DQ chemical shifts are scaled by 1/2

Where does the additional increase in resolution (> 2) come from?

See Vega, Pines (1977) J. Chem. Phys. 66 5624

€

HQ =ωQ

33Iz Iz − I ⋅ I[ ]

ωQ =e2qQ

2I(2I −1)12 (3cos2θ −1)[ ] ; η = 0

2H-DQ are insensitive to MA mis-settingand to Motional Broadening

Simulated 2H off-Magic Angle spectra

CQ = 100 kHz MAS = 20 kHz

Setting the Magic Angle using NaNO3

2H spectroscopy applied to α-spectrin SH3

13C detected 1H,13C CP

Dα-Cα

Dmet-Cmet

2H-DQ,13C CP

2H spectroscopy applied to α-spectrin SH3: Methyl spectral region13C detected 1H,13C CP 2H-DQ,13C CP

S/N(2H,13C) ≈ 2-3 x S/N(1H,13C)

γ(1H) = 6.5 x γ(2H) T1 (1H) = 4 x T1 (2H) n(2H) = 33-36 x n(1H)

2H spectroscopy applied to α-spectrin SH3

Cα-Dα

Agarwal, Faelber, Schmieder, Reif, JACS 131, 2 (2009)

Can we detect aliphatic protons ? (other than methyls)

Can we detect aliphatic protons (other than methyls) ?

Biosynthesis with 2H,13C glucose and various amounts of H2O (5-30 %)

NOESY type distance restraints in the solid-state

3. Paramagnetic

Wickramasinghe, Kotecha, Samoson, Past, Ishii J. Magn. Reson. 184, 350 (2007)Linser, Diehl, Chevelkov, Reif J. Magn. Reson. 189, 209 (2007)

0 mM Cu-EDTA 75 mM Cu-EDTA

Gain: Line width: x4 Proton detection: x9

Loss:

Dilution: x10

Doping with Cu(II)-EDTAHow can we do better ?

http://www2.warwick.ac.uk/fac/sci/physics/

Perspectives:

1H detection using protonated samples

in the solid-state at very high MAS

frequencies

u-[2H,15N]-GB1, 39 kHz MAS, 750 MHz: Back-exchanged with 100 % H2O

Zhou, Shea, Nieuwkoop, Franks, Wylie, Mullen, Sandoz, Rienstra (2007) Angewandte Chemie Int. Edt. 46 8380-8383

Faster is better ….

Lewandowski, Dumez, Akbey, Lange, Emsley, Oschkinat (2011) J. Phys. Chem. Lett. 2 2205-2211

u-[2H,15N] α-SH3 re-crystallized with 100 % H2O 60 kHz MAS, 1000 MHz

MAS rotor Volume

max concentration exchangeable

protonscoil

efficiencyrelative intensity

1.3 mm 4 µL 100 % 2.5 1.25

3.2 mm 40 µL ca. 20 % 1 1

Akbey, Lange, Franks, Linser, Diehl, van Rossum, Reif, Oschkinat, JBNMR 46, 67-73 (2010) Lewandowski, Dumez, Akbey, Lange, Emsley, Oschkinat J. Phys. Chem. Lett. 2 2205-2211 (2011)

Increase of the MAS frequency allows to use higher proton densities and results in a reduction of the sample mass

u-[2H,15N] α-SH3 100 % H2O MAS = 60 kHz

u-[2H,15N] α-SH3 10 % H2O MAS = 60 kHz

A55

u-[2H,15N] α-SH3 differing [H2O] @ crystallization MAS = 20 kHz

Proton detection at FAST MAS with Protonated Protein Samples

1H detection of protonated proteins

Zhou, Shah, Cormos, Mullen, Sandoz, Rienstra (2007) J. Am. Chem. Soc. 129 11791-11801; Marchetti, Jehle, Felletti, Knight, Wang, Xu, Park, Otting, Lesage, Emsley, Dixon, Pintacuda (2012) Angewandte Chemie Int. Edt. 51 10756-10759; Andreas, Jaudzems, Stanek, Lalli, Bertarello, Marchand, Cala-De Paepe, Kotelovica, Akopjana, Knott, Wegner, Engelke, Lesage, Emsley, Tars, Herrmann, Pintacuda, Proc. Natl. Acad. Sci. U.S.A. 113: 9187-9192 (2016)

from Pintacuda and co-worker

Structural analysis of the protonated Acinetobacter phage 205 coat protein (AP205CP)

Andreas, Jaudzems, Stanek, Lalli, Bertarello, Marchand, Cala-De Paepe, Kotelovica, Akopjana, Knott, Wegner, Engelke, Lesage, Emsley, Tars, Herrmann, Pintacuda, Proc. Natl. Acad. Sci. U.S.A. 113, 9187-9192 (2016)

Marchetti et al. (2012) Angewandte Chemie Int. Edt. 51, 10756-10759

Proton detected experiments of Protonated and Deuterated Protein Samples

Lewandowski et al. (2011) J. Phys. Chem. Lett. 2 2205-2211

u-[2H,15N] α-SH3, 1000 MHz

u-[1H,15N] SSB, 800 MHz

1H dipolar interactions yield residual broadening in protonated α-SH3 at 110 kHz

u-[1H,13C] α-SH325 % RAP-[2H,13C] α-SH3

Xue, Sarkar, Motz, Rodriguez Camargo, Decker, Wegner, Asami, Tosner, Reif, Sci Rep 7, e7444 (2017)

Applications to non-crystalline systems

MAS induces reversible sedimentation of protein complexes

Bertini et al., PNAS 108, 10396 (2011)

Challenges in the Solid-State: - Crystal contacts - Ligands have to be co-precipitated - Analysis of chemical shifts is ambiguous if crystal symmetry changes

Challenges in Solution-State: - Correlation time problem: Resonance Lines become broad for large molecules - TROSY (Pervushin/Wüthrich) - Decrease of τC: High temperature / Reversed Micelles (Wand)

αB inhibits amorphous aggregation of reduced lysozyme

Soluble Protein Complexes: The proteasome activator complex 11S-α7(β7β7)α7 [Thermoplasma acidophilum]

11S-α7β7β7α7 1.1 MDa

perdeuterated, back-exchanged with 20% H2O; 20 kHz MAS, 600 MHz

Mainz et al., Angewandte Chemie Int. Edt. 52 8746-8751 (2013)

Ribosomal Complexes: How far can we go ?

in collaboration with Shang-Te Danny Hsu, Taiwan and Roland Beckmann, LMU München

Questions:

• Can we observe individual components of the ribosome ?

• Does ribosome binding induce a conformational change of Trigger Factor (TF) ?

Barbet-Massin, Angewandte Chemie Int. Edt. 54, 4367 (2015)

Perspectives for Larger Protein Complexes

Sedimented protein fractionriri

Mw

Distance from rotor axis at which max. concentration is reachedωRρsolv

T

tightest packing of spheres

100% exchangeable protons: x 5 Q-factor (1.3/3.2 mm): x 2.5 MAS (60 kHz): x 1/10 (0.2 mM = 2 mg in 4 µL)

theoretical S/N (HSQC) = 2.5-7.5 : 1 (EXPT = 12 h)

Proteasome: 11S14-α7(β7β7)α7 (1.1 MDa) c limit = ~ 1.0 mM (particle) ------------------------------------- 3.0 mM (α-monomer) 0.2 mM (particle conc.) 4.6 mg in 20 µL 3.2 mm MAS, 20 % H2O S/N (HSQC) = ca. 30-90 : 1 (EXPT = 12 h)

for αB-crystallin (24-mer, 20 kDa): c limit = ~53 mM (monomer)

prokaryotic ribosome (2.5 MDa): c limit = ~ 0.4 mM

TF-RBD in complex with the 50S ribosome

solution-state (TF-RBD)

in complex with 50S

unbound TF-RBD in solution

TF-RBD in complex with 50S

TF-RBD in complex with the 50S ribosome

Baram et al. Yonath PNAS 102 12017 (2005)

no chemical shift changes chemical shift changes / exchange broadening

TF (Ribosome Binding Domain) (~14 kDa) in complex with 50S (~1.4 MDa) 100 % back-exchanged @ 60 kHz MAS: ca. 20 µg TF-RBD

solution-state (TF-RBD) in complex with 50S

Processing of the Amyloid Precursor Protein (APP) yields the Alzheimer's disease β-amyloid peptide Aβ

Solid-State NMR of Aβ aggregates

Lansbury et al. Griffin, Nat. Struct.Biol. 2, 990 (1995) Benzinger et al. Meredith, Proc. Natl. Acad. Sci. USA 95, 13407 (1998) Petkova et al. Tycko, Science 307, 262 (2005) Petkova et al. Tycko, Biochemistry 45, 498 (2006) Paravastu, et al. Tycko, Proc. Natl Acad. Sci. USA 106, 7443 (2009) Bertini et al. Mao J. Am. Chem. Soc. 133, 16013 (2011)

Chimon et al. Ishii, Nature Stuct. Mol. Biol. 14, 1157 (2007) Ahmed et al. Smith, Nature Struct. Mol. Biol. 17 561 (2010)

Amyloids: Alzheimer’s disease Aβ40 fibrils

Linser et al., Angewandte Chem. Int. Edt. 50, 4508-4512 (2011)

Tex50 nm

• 1 set of resonances (30-40 peaks) • Well defined chemical shift dispersion → well-defined 3D structure

3D-HNCO

Alzheimer's disease Amyloid Aβ(1-40) 1H,15N CP correlation (full spectrum)

Histidine imidazole correlations ?

Lys-28 ?

Alzheimer's disease Amyloid Aβ(1-40) 1H,13C CP correlation

H2O

Ser

His

Detection of Histidines Imidazole Protons in Amyloid Aβ(1-40) fibrils

Detection of Histidines Imidazole Protons in Amyloid Aβ(1-40) fibrils

Hδ1

-

Detection of Histidines Imidazole Protons in Alzheimer's disease Amyloid Aβ(1-40) fibrils

Hδ1

H13

H14 K16

Q15

E22

E11

Tycko, Quart Rev Biophys. 39 1-55 (2006)

Cα Gln-Cβ/ Glu-Cβ

Exchangeable side chains can assist in determining the quarternary structure of amyloid fibrils

Histidines can assist in determining the quarternary structure of Amyloid Aβ(1-40) fibrils

Hδ1

Agarwal et al., PCCP 15, 12551 (2013); see also: Petkova et al. PNAS 99 16742 (2002)

Membrane proteins: OmpG and Bacteriorhodopsin

Bacteriorhodopsin

OmpG

Linser et al. Angewandte Chemie Int. Edt. 50, 4508-4512 (2011)

Protein-RNA Complexes: The boxCD - L7ae complexof Archeoglobus fulgidus

Asami et al. Angewandte Chemie Int. Edt. 52 2345-2349 (2013)

solution-state solid-state

HN correlations of the boxCD RNA-Protein Complex

Superposition of the Solid-State and Solution-State Spectra

solution-state solid-state

Protein HN correlation spectra in presence of protonated and deuterated RNA

Other examples: Proton detection in non-crystalline systems

Ward, M.E., Shi, L., Lake, E., Krishnamurthy, S., Hutchins, H., Brown, L.S., and Ladizhansky, V. (2011). Proton-Detected Solid-State NMR Reveals Intramembrane Polar Networks in a Seven-Helical Transmembrane Protein Proteorhodopsin. J Am Chem Soc 133, 17434-17443.

Chevelkov, V., Habenstein, B., Loquet, A., Giller, K., Becker, S., and Lange, A. (2014). Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins. J Magn Reson 242, 180-188.

Eddy, M.T., Su, Y.C., Silvers, R., Andreas, L., Clark, L., Wagner, G., Pintacuda, G., Emsley, L., and Griffin, R.G. (2015). Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR. J Biomol NMR 61, 299-310.

Andreas, L.B., Reese, M., Eddy, M.T., Gelev, V., Ni, Q.Z., Miller, E.A., Emsley, L., Pintacuda, G., Chou, J.J., and Griffin, R.G. (2015). Structure and Mechanism of the Influenza A M2(18-60) Dimer of Dimers. J Am Chem Soc 137, 14877–14886.

Acknowledgement

Vipin Agarwal Sam Asami Veniamin Chevelkov Rasmus Linser

Purdue University Nikolai Skrynnikov