Magic-Angle Spinning (MAS) drive bearing

Transcription

1 Magic-Angle Spinning (MAS) magic-angle spinning is done pneumatically spinning frequency can be stabilized within a few Hz Magic-Angle Spinning (MAS) drive bearing

2 Magic-Angle Spinning (MAS) Maximum spinning frequency depends on rotor diameter some typical diameters: 4.0 mm 15 khz (1,400,000 x g) 3.2 mm 25 khz (2,700,000 x g) 2.5 mm 35 khz (3,500,000 x g) (50,000 x g) Solid-state MR is brute force

3 Barth-Jan van Rossum: Solid-State MR Magic-Angle Spinning (MAS) a 3.2 mm rotor spinning at 24 khz has a speed of 240 m/s when it would roll on the floor and needs only 46 hours to roll around the earth

4 Chemical shift anisotropy (CSA) σ iso = ( σ 11 + σ 22 + σ 33 ) / 3 σ iso = isotropic chemical shift - is what remains for very fast MAS - is the shift as detected in liquid-state MR the isotropic shift does generally not coincide with maximum of the powder line shape σ iso σ iso static σ iso magic-angle spinning spherical symmetry non-axial symmetry axial symmetry

5 Isotropic chemical shift chemical shifts of a protein determined by: - folding of the protein - interactions between proteins (solids!) - environmental factors (ph, temperature,solvent ) protein surface: shift differences in contact areas protein core: shift differences less likely there is no such thing as the MR chemical shift!!

6 Aggregation shifts shifts for P20 CHδ: Hδ = -1.5 ppm Cδ = -1.1 ppm SH3 I Tyr Å δ 3.5 Å Ser 19 Pro 20 SH3 I SH3 II Phe Å 4.3 Å δ1 Solid state shift (ppm) E7 L8 K18 S19 P20 E22 I30 P54 SH3 II Leu 8 shifts for L8 CHδ1: Hδ1 = -1.3 ppm Cδ1 = -2.0 ppm Liquid state shift (ppm)

7 MAS: spinning side bands What spinning frequency to use? 1. large enough to have most intensity in the centre band (isotropic shift) 2. remaining side bands should not interfere with other signals example: spectrum recorded at 700 MHz, with 12 khz MAS 12,000 Hz * 30,000 25,000 20,000 15,000 10,000 5, ,000 Hz 700 MHz 1 ppm = 700 Hz ( 1 H) 1 ppm = 175 Hz ( 13 C) γ H : γ C = 4 : 1 12,000 Hz (MAS) ~ 69 ppm (13C)

8 MAS: spinning side bands Care should be taken when choosing the MAS frequency to avoid overlap and rotational resonance In 2D spectra, side-bands appear as additional diagonal patterns 10,000 Hz side bands diagonals in 2D spectra are not artefacts 10,000 Hz (2D spectrum recorded at 750 MHz, with 10 khz MAS)

9 MAS: spinning side bands Care should be taken when choosing the MAS frequency to avoid overlap and rotational resonance more tricky: side bands of correlation signals! 13,000 Hz 13,000 Hz 13,000 Hz (2D spectrum recorded at 900 MHz, with 13 khz MAS)

10 MAS: spinning side bands Care should be taken when choosing the MAS frequency to avoid overlap and rotational resonance MAS frequency matches the chemical shift difference of two signals (in Hz) signals are coupled due to rotational resonance effect (line splitting!) should generally be avoided C 600 MHz 1 ppm = 150 Hz ( 13 C) 18 khz (MAS) 120 ppm ( 13 C) Cα 18,000 Hz 25,000 20,000 15,000 10,000 5, ,000 Hz Rotational resonance

11 The CP-MAS experiment the standard MAS MR experiment to detect 13 C 1 H CP 13 C CP decoupling 1 H used to enhance 13 C signal via cross-polarization (CP) 1 H removed during data acquisition (decoupling) 1 H only used to get more signal

12 Effect of CP and decoupling on sensitivity 1 H CP decoupling 13 C CP CP + decoupling CP (no decoupling) (no CP) decoupling (no CP, no decoupling)

13 Effect of MAS and decoupling on 13 C resolution no MAS, no decoupling no MAS, decoupling MAS, no decoupling MAS, decoupling solution MR

14 Chemical shift assignment: identification of side chains assignment procedure is a two-step mechanism 1. identification of spin-systems (amino acids) 2. connecting spin-systems via backbone γ L-threonine β α C' Cγ Cβ Cα T C' β α γ L Cα Cβ C' Cα Cβ I Cγ Cγ1 Cγ2 Cδ1 Cδ1 Cδ γ C chemical shifts are used to identify spin systems (in liquids 1 H shifts are used for identification) 80 β 60 α

15 Chemical shift assignment: identification of side chains residues can be identified from characteristic 13 C- 13 C correlation patterns β α γ α β threonine γ 20 β 20 alanine γ α β β α α α β β α α β γ1, γ serine β α 20 γ1, γ2 20 β β α 60 α valine γ1 β γ2 α

16 Chemical shift assignment: sequential assignment specific or adiabatic CP typical building blocks for finding sequential connectivities C CA C transfer: inter-residual, connects sequential residues CA transfer: intra-residual, transfer within residue

17 Sequential assignment Cγ Cβ Sequential assignment of L131-T132-I133 motive L Cα Cβ Cγ Cδ1 Cδ1 C' Cα T C' I Cα C' Cβ Cγ1 Cγ2 Cδ L131 (115.6) T132 (106.4) I133 (121.8) CACX 1,3- αbc CACX U- αbc CCX U- αbc CACX U- αbc CCX U- αbc CACB U- αbc

18 Sequential assignment Cγ Cβ Sequential assignment of L131-T132-I133 motive L Cα Cβ Cγ Cδ1 Cδ1 C' Cα T C' I Cα C' Cβ Cγ1 Cγ2 Cδ L131 (115.6) T132 (106.4) I133 (121.8) CACX 1,3- αbc CACX U- αbc CCX U- αbc CACX U- αbc CCX U- αbc CACB U- αbc

19 Dipolar interaction B θ the dipolar coupling is the interaction between two magnetic moments it depends on : r - the distance between the spins (r) - the angle θ between the magnetic field B and the vector connecting the spins D 1 r 3 ( 3cos 2 θ 1) 3cos 2 θ 1 = 0 in isotropic liquids 0 in solids or oriented media

20 Dipolar interaction under magic angle spinning, the average angle between the connection vector and B is the magic angle θ m B θ m θ m θ 3cos 2 θ 1 0 3cos 2 θ 1 1 m = 0

21 Dipolar interaction The time average of every spin-pair has the connecting vector at the magic-angle time average is zero for θ = cos 2 θ 1 1 m = 0 θ = 54.7

22 Interaction under MAS Similar considerations can be made for any interaction that contains the 3cos 2 θ 1 term, like the chemical shift interaction Chemical shift interaction Dipolar interaction anisotropic part + isotropic part anisotropic part 3cos 2 θ 1 not orientation dependent 3cos 2 θ 1 MAS

23 Dipolar interaction ote: the time average is zero, not the dipolar coupling itself the dipolar interaction is time dependent and oscillates around 0 θ m

24 Recoupling The time-average of the dipolar coupling is zero but not the instantaneous coupling, it is oscillating around zero

25 Recoupling Cγ recoupling techniques are useful for short-range transfer (e.g., for assignment of spin systems) L Cα Cβ Cγ C' Cβ Cα T C' C' Cα Cβ Cγ1 Cγ2 I Cδ1 Cδ1 Cδ D 1 r 3 ( 3cos 2 θ 1) however, distance information is more difficult to obtain if more than two spins interact in particular, for uniform isotope labelling, long range transfer is difficult due to dipolar truncation effects

26 Barth-Jan van Rossum: Solid-State MR Dipolar truncation the phenomenon that weak couplings are quenched by the strong couplings à weak couplings contain the long-range distance information 1 2 D 3 (3cos θ 1) r The layman s view on dipolar truncation oops strong coupling weak coupling

27 Dipolar truncation the phenomenon that weak couplings are quenched by the strong couplings weak couplings contain the long-range distance information spin dilution D 1 r 3 ( 3cos 2 θ 1) note: pair-wise labelling is maximal spin dilution

28 Dipolar truncation the phenomenon that weak couplings are quenched by the strong couplings weak couplings contain the long-range distance information spin dilution selective recoupling D 1 r 3 ( 3cos 2 θ 1)

29 Extensive 13 C labelling to reduce dipolar truncation amino-acid labelling bacteria grown on: 1,3-13 C glycerol (green) or 2-13 C glycerol (red) labelling mostly alternating strong couplings removed weak couplings not quenched G H S W C F A Y V L R Group I H2 H2 H2 H2 H H H H D M T I K H2 Group II R R1 (Pro) R2 H2 H2 H2 H2 H H H H however, analytical treatment will still be extremely difficult

30 Barth-Jan van Rossum: Solid-State MR Extensive 13C labelling to reduce dipolar truncation T37 Cβ β T24 Cβ T32 C à less signals in the spectra I30 Cδ à partial suppression of J-couplings [U -13C] à less assignment options ppm T37 Cβ T32 Cβ T24 Cβ ppm ppm I30 Cδ [2-13C] H2 H SW R CF Y A H H2 H (Pro) D M T H2 R1 R2 H2 ppm ppm H 180 H T37 Cβ T32 Cβ T24 Cβ I30 Cδ ppm [1,3-13C] I V H2 H H2 H K H2 L Group I R G H2 H Group II H2 ppm ppm H ppm