Longitudinal-relaxation enhanced fast-pulsing techniques: New tools for biomolecular NMR spectroscopy Bernhard Brutscher Laboratoire de Résonance Magnétique Nucléaire Institut de Biologie Structurale - Jean Pierre Ebel Grenoble, France IBS TGE-RMN 1 st Users meeting, Paris, October 1 st, 2009
Multidimensional NMR spectroscopy 1D 1 H 2D 1 H 15 N 3D 15 N 13 C 1 H 15 N 1 H Site-resolved information on molecular structure & dynamics
Major limitations of multidimensional NMR Low experimental sensitivity Long experimental times t 1 mix t 2 mix t acq Recycle delay B 0 time scale: 1-2 seconds 10 17 molecules t 2 - Only 1 out of ~10000 molecules contributes to NMR signal. Data sampling grid - Signal loss during various coherence transfer and frequency editing steps. t 1 1D 2D 3D 4D 5D seconds minutes hours weeks years Increase sensitivity Decrease experimental time
Polarization-enhanced fast-pulsing techniques BEST Gain in sensitivity Gain in speed = reduced acquisition time
Longitudinal spin relaxation and NMR sensitivity wait... ~0.1 s 1-2 s scan time z-magnetization Longitudinal relaxation 0 1 2 3 Sensitivity S/N S/N ~ (number of scans) ~1/ (scan time) 1 2 scan time / seconds Enhanced longitudinal 1 relaxation: Shorter optimal Scan repetition time [sec] rates increased experimental sensitivity 0 2 3
Longitudinal 1 H relaxation in macromolecules Solomon equations: I 1z -I 1z 0 Σ j ρ 1j σ 12 σ 13... σ 1n I 1z -I 1z 0 d dt I 2z -I 2z 0... I nz -I nz 0 = σ 21 Σ j ρ 2j σ 23... σ 2n... σ n1 σ n2 σ n3 Σ j ρ nj I 2z -I 2z 0... I nz -I nz 0 The spin-lattice relaxation of a 1 H spin depends on the initial spin-state of the surrounding 1 H. (noe or spin diffusion effect) In addition: chemical exchange with water 1 H
Longitudinal amide 1 H relaxation enhancement Idea: excite only a small number of protons, e. g. amide 1 H Aliphatic 1 H saturated ( ) H N H α H M z = 0 H H M z = 0 M z = 0 0 1 2 0 1 2 Recovery time Recovery time [sec] [sec] Aliphatic 1 H in equilibrium ( ) H M z = M eq H M z = 0 H M z = M eq H N H α 0 1 2 0 1 2 Recovery time Recovery time [sec] [sec]
Longitudinal amide 1 H relaxation enhancement Magnetic field (B 0 ) dependence selective non-selective non-selective selective
Further optimization of steady-state proton polarization: Ernst-angle excitation H α HMQC-1: α opt =180 -α 90 α opt 60 40 Ernst-angle excitation HMQC-2: α opt =2πJ HX Δ 20 cos α opt = exp(- t rec / T 1 ) 0.5 1.0 1.5 2.0 2.5 t rec / T 1
Longitudinal spin relaxation, excitation angle, and NMR sensitivity " $ # S N % ' & t = f ( T rec,t scan,t 1,() ) ( 1* exp (*T rec T 1 ))sin( 1* exp (*T rec T 1 )cos( ( ) T scan! T scan (i) T scan (i+1) 1.0 45 90 BEST t seq t acq t rec 0.5 45 90 T rec 0 0 0.5 1.0 1.5 2.0 2.5 3.0 Recovery time T rec (s)
Sensitivity gain from BEST/SOFAST experiments high-speed and optimal-sensitivity regime Sensitivity [a.u.] 4 3 2 1 ~ 30-100% more sensitive 0 0 1 2 Recovery time [sec] Sensitivity gain of a factor of 5-10
BEST-type experiments Band-Selective Excitation Short-Transient Experiments aliphatic amide 10 8 6 4 2 0 ppm BEST Example: HNCO and HNCA standard band-selective pulses broadband inversion pulses
BEST 1 H- 15 N HSQC 8.6 kda 600 MHz data 12 kda BEST 1 H- 15 N TROSY 21 kda
BEST-type experiments available (so far) 2D BEST-HSQC, 2D BEST-TROSY (Schanda et al., JACS (128) 2006, 9042; Farjon et al., JACS (131) 2009, 8571) Sample quality control, titrations, chemical shift mapping 3D & 4D BEST-HNC triple resonance experiments (Schanda et al., JACS (128) 2006, 9042; Lescop et al., JMR (187) 2007, 163) Sequential resonance assignment, backbone RDCs BEST-TROSY-HNN (Farjon et al., JACS (131) 2009, 8571) Trans-H-Bond couplings in RNA/DNA BEST-HMQC2 (Schanda et al., J Biomol NMR (38) 2007, 47) Measurement of H N -H N RDCs BEST- 15 N-relaxation dispersion and BEST-EXSY experiments (Kern et al., unpublished) Conformational exchange
The SOFAST-HMQC experiment Combining the advantages of Longitudinal-relaxation enhancement and Ernst-angle excitation! 6 H N G z β > 90 180 Band-Selective Optimized- Flip-Angle Short-Transient Sensitivity [a.u.] 4 2 0 standard se-hsqc SOFAST 90 SOFAST 120 SOFAST 150 0 1 2 scan time [sec] t rec = 0-100 ms
Applications of SOFAST-HMQC Ubiquitin (8.6 kda, 0.2 mm) trna Val (26 kda kda, 0.9 mm) 4 sec. 3 sec. TET-2 (486 kda, 80 µm, ~ 1 mm protomer concentration) 3.4 sec.
Investigating the protein folding energy landscape by real-time 2D NMR
SOFAST real-time 2D NMR 3 2 1 0 Time [s] 0 1 2 3 4 time [sec] 4
SOFAST real-time 2D NMR Initiation of kinetic event 12 8 4 0 Time [s] 16
β2-microglobulin: folding of an amyloidogenic protein Unfolded Intermediate(s)? Folded Fibrils ph jump (Dialysis-related amyloidosis) Unfolded, ph 2 t=0 seconds Final spectrum U I1 N
0 sec 15 sec 30 sec 45 sec 105 sec 165 sec 225 sec 325 sec 420 sec final I I N N H N 95 H N 28 Disappearance of I-state peaks Appearance of native state peaks (I+N) kinetics mono-exponential bi-exponential
Unfolded (~90%) (~10%) k > 0.2 s -1 Intermediate I1 (NMR visible) k > 0.2 s -1 k 0.002 s -1? Intermediate I2 (NMR invisible) Native (cis-pro32) 1.0 (I+N) kinetics P32 (trans in I1) No chemical shift changes between N-state and I1-state 0.8 0.6 0.75 mm 0.38 mm 0.18 mm 0 1000 2000 3000 t (s)