assignments for uniformly-labeled proteins 2. Biomolecular solid state NMR at low and ultra-low temperatures

Transcription

1 Lecture by R. Tycko at 3 rd Winter School on Biomolecular Solid State t NMR Stowe, Vermont, January 8, 2013 Two topics: 1. Computer-aided NMR resonance assignments for uniformly-labeled proteins 2. Biomolecular solid state NMR at low and ultra-low temperatures

2 Computer-aided resonance assignments for uniformly 15N,13C-labeled proteins (Tycko and Hu, JMR 2010; Hu et al., JBNMR 2011) connections -N-CA-CO-N-CA-CO-N-CA-CO-N-CA-CO- CX CX CX CX 2D (or 3D) NCACX 2D (or 3D) NCOCX Manual assignment succeeds when signal-to-noise is high, lines are sharp (especially 15N), and residue-type assignments (based on CX signals) are unambiguous. Otherwise manual assignment is tedious, error-prone, and incomplete. U-labeled HET-s( ) prion fibrils (see papers by B. Meier and coworkers)

3 Computer-aided resonance assignments for uniformly 15N,13C-labeled proteins NCACX Signal-to-noise is low. Resolution is good, but not excellent. NCOCX 208-residue protein, but only ~35 residues contribute to solid state NMR spectra. Can we get any useful information from such spectra? U-labeled mammalian PrP fibrils (with I.V. Baskakov)

4 Computer-aided resonance assignments for uniformly 15N,13C-labeled proteins Strategy: Humans are good at this. Manually create tables of crosspeak positions (i.e., NMR frequencies) and possible residue-type assignments (which can be ambiguous) for each 2D/3D spectrum. Use a Monte Carlo/simulated annealing algorithm to assign crosspeaks to specific residues in the protein sequence so that t good connections are maximized, bad connections are eliminated, all signals are used, and edges are minimized. Run this algorithm many times to identify all assignments that are consistent with the available data. Computers are good at this.

5 Computer-aided resonance assignments for uniformly 15N,13C-labeled proteins Results from 100 MC/SA assignment runs on U-labeled PrP fibril data Limited information, but better than nothing. Accurate representation of the information content of the NMR data. (Tycko et al., Biochemistry 2010)

6 INPUTS: Generalized MCSA algorithm for arbitrary combinations of multidimensional spectra (Hu et al., JBNMR 2011) Lists of multidimensional signals from any 2D/3D/4D spectra, containing chemical shifts, uncertainties in chemical shifts, and possible residue-type assignments (e.g., EQKRWH or DN or S23). Table of connections among pairs of signals in pairs of spectra. Which chemical shifts should agree if the sequential assignment is correct? Protein sequence (arbitrarily-defined one-character symbols; applicable to any biopolymer, allows >> 20 residue types ) Score = w 1 N G -w 2 N B -w 3 N E + w 4 N U Starting ti with "null assignment", randomly change assignments of signals to residues (one at a time) and accept/reject changes based on Metropolis criterion: e S > ran[0,1]. Gradually increase w 1, w 2, w 3, and w 4 from zero to their maximum values. Run many times, inspect final assignments with N B = 0 to determine uniqueness. Well-defined, reproducible process. Assumptions regarding data are displayed explicitly in the input files. Finds ALL assignments that are consistent with the data, not just one. Identifies segments with unique assignments, multiple l assignments, no assignments, etc. Provides a guide for additional measurements, labeling schemes, etc.

7 Recent example: 40-residue -amyloid fibrils derived from human brain tissue

8 Why NMR at low (<200 K) or ultra-low temperatures t (<77 K)? Study (non-biological) phenomena that occur only at very low temperatures. t Rotational diffusion is suppressed in frozen solutions, allowing solid state t NMR techniques to be employed. Trap transient structural or chemical states. Enhance sensitivity.

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10 orientational order/disorder transition

11 electron energy AlGaAs aas Ga AlGaAs

12 AlGaAs contact hyperfine shift, proportional to electron spin polarization in quantum wells GaAs Maximum electron spin polarization only when lowest level is exactly filled. Adding or removing one electron causes several other electrons to flip.

13 How does one do such experiments? What does the NMR probe look like? helium transfer line vacuum can copper block, controlled temperature radiation shield

14 How does one do such experiments? What does the NMR probe look like? Janis SuperTran cryostat (ST-200-NMR)

15 Why NMR at low (<200 K) or ultra-low temperatures (<77 K)? Study (non-biological) phenomena that occur only at very low temperatures. t Rotational diffusion is suppressed in frozen solutions, allowing solid state t NMR techniques to be employed. Trap transient structural or chemical states. Enhance sensitivity.

16 V3 peptide, 13C-labeled at two backbone carbonyl sites V3/antibody Fab complex Nature Struct. Biol DQ-filtered MAS at 150 K, using Chemagnetics probe, 240 l sample, 4 mm peptide/antibody, frozen solution doped with Cu-EDTA, long experiments

17 JACS 2004 free peptide antibody-bound bound peptide

18 Why NMR at low (<200 K) or ultra-low temperatures (<77 K)? Study (non-biological) phenomena that occur only at very low temperatures. t Rotational diffusion is suppressed in frozen solutions, allowing solid state t NMR techniques to be employed. Trap transient structural or chemical states. Enhance sensitivity.

19 Protein folding: What can solid state NMR contribute? folded state (relatively rigid, unfolded state (dynamic, relatively compact, highly ordered) extended, highly disordered) temperature, ph, denaturant What are the conformational distributions in the unfolded state of a simple protein? (site-specific and quantitative) How do conformational distributions vary as the equilibrium shifts from the folded to the unfolded state? (see K.-N. Hu et al., JMB 2009) Can we trap intermediate states along the folding pathway and examine their structures in detail by solid state NMR?

20 Val50 Leu69 villin HP35 Experiments by Eaton, Raleigh,... Simulations by Pande Schulten Shaw Shakhnovich Simulations by Pande, Schulten, Shaw, Shakhnovich,... Supposed to fold on the 5 s time scale.

21 Rapid freeze-trapping of transient structural states heated tube, containing ~200 l of protein solution in glycerol/water HPLC pump to expel protein solution from tube miscellaneous parts for separating frozen protein solution from isopentante, Kan-Nian packing Hu into MAS rotor for solid state NMR 20 m-diameter aperture at end of tube, creates 20 m- diameter jet, 10 4 cm/s isopentane bath at 140 K, freezes ~10 m-diameter droplets in s

22 Rapid freeze-trapping of transient structural states HP35, U- 13 C-labeled Val50, 13 CO, 13 C -labeled Leu69. glycerol/water solution. DQ-filtered 13 C NMR at 140 K VLCO V,L L C splitting of V C Val50 methyl signals V C V C Rapidly frozen from room temperature Rapidly frozen from +90 C (folded state only) (apparently folded/unfolded mixture)

23 HP35, U- 13 C-labeled Val50, 13 CO, 13 C -labeled Leu69. Glycerol/water solution. Two-dimensional 13 C NMR at 140 K fully folded state No detectable fully-folded folded HP35 molecules in our freeze-quenched samples. (Rather surprising) Val50 C /C crosspeak rapidly frozen from +90 C unfolded component folded component Val50 C /C crosspeak

24 Additional evidence for an intermediate with incomplete tertiary structure (JACS 2010) U-labeled A49, G52, M53, F58, P62, and L69 slowly frozen H3 H1 freeze-quenched H2 Met53 and Gly52 signals are broad, implying a disordered loop between H1 and H2.

25 Additional evidence for an intermediate with incomplete tertiary structure (JACS 2010) slowly frozen freeze quenched 13 C spin polarization transfers from Phe58 to Leu69 and Met53 are supressed, implying disruption of the hydrophobic core.

26 HP35, U- 13 C-labeled Val50, 13 CO, 13 C -labeled Leu69. Glycerol/water solution. Two-dimensional 13 C NMR at 140 K fully folded state One week of continuous signal averaging, 5 mm protein, 200 l volume rapidly frozen from +90 C

27 Why NMR at low (<200 K) or ultra-low temperatures (<77 K)? Study (non-biological) phenomena that occur only at very low temperatures. t Rotational diffusion is suppressed in frozen solutions, allowing solid state t NMR techniques to be employed. Trap transient structural or chemical states. Enhance sensitivity.

28 Sensitivity is often the main limitation on NMR measurements in general, and biomolecular solid state NMR in particular. We usually need 1-20 mg of isotopically i lbld labeled protein, ti and days for each 2D or 3D spectrum. NMR signal amplitudes are proportional to nuclear spin polarizations (difference between "up" and "down" spin state populations in a magnetic field). Nuclear spin polarizations are very small (~10-5 ), and proportional to 1/T at thermal equilibrium i signals can be enhanced 12X by cooling the sample from 300 K to 25 K. (but we need magic-angle spinning)

29 Ultra-low-temperature MAS probe -- Helium used for cooling only -- Nitrogen used for bearing and drive -- Long rotors to separate cold sample volume from warmer rotor ends -- Internal baffle to keep nitrogen gas out of the sample area

30 Ultra-low-temperature MAS probe sodium acetate in glycerol/water, 200 M DyEDTA 7.00 khz MAS, 1.5 ms CP, 105 khz decoupling Ac-HQKLVFFAED-NH 2 H 2 N-DEAFFVLKQH-Ac Ac-HQKLVFFAED-NH 2 H 2 N-DEAFFVLKQH-Ac A fibrils, U-labeled A21 and V mg (2 mole) in glycerol/water 6.7 khz MAS 75 khz TPPM decoupling in t1 and t2 3.5 hr exp t, 6 s recycle delay, 1.2 ms RFDR mixing (100 khz decoupling) 500 ms spin diffusion

31 Ultra-low-temperature MAS probe sodium acetate in glycerol/water, 200 M DyEDTA 7.00 khz MAS, 1.5 ms CP, 105 khz decoupling Ac-HQKLVFFAED-NH 2 H 2 N-DEAFFVLKQH-Ac Ac-HQKLVFFAED-NH 2 H 2 N-DEAFFVLKQH-Ac A fibrils, U-labeled A21 and V mg (2 mole) in glycerol/water 6.7 khz MAS 75 khz TPPM decoupling in t1 and t2 3.5 hr exp t, 6 s recycle delay, At low temperatures, nuclear spin polarizations can be further enhanced by driving them out of thermal equilibrium, through cross-relaxation processes involving out-of-equilibrium electron spins. This is called "dynamic nuclear polarization", or DNP. See experiments by Griffin group at MIT since ms RFDR mixing (100 khz decoupling) 500 ms spin diffusion

32 detector for EPR tunable 264 GHz microwave source (Virginia Diodes, Inc.) quasi-optical polarizer (Thomas Keating, Ltd.) cryostat head with modulation coil (Janis Supertran)

33 1 H solid echo, glycerol/water at 11 K, 30 mm DOTOPA-TEMPO with waves DNP build-up time without waves net sensitivity gain 1600X 250X relative to room temp

34 How does DNP work? net transfer from n up to n down energy-conserving flips of all three spins due to their couplings microwave irradiation at e1 frequency two electrons, one nucleus, all coupled to one another "cross effect" mechanism for DNP in a static sample, when difference in electron spin flip energies equals the nuclear spin flip energy.

35 How does DNP work under MAS?

36 How does DNP work under MAS? Most (but not all) biradicals contribute to DNP. (Triradicals are better?) DNP enhancement increases with DNP enhancement increases with increasing microwave power, up to a point. Longer T 1e implies lower microwave power.

37 Ultra-low-temperature MAS/DNP probe (Thurber et al., J. Magn. Reson. 2013)

38 9.4 T, 89 mm bore Oxford magnet microwave system

39 DNP-enhanced solid state NMR with MAS at very low temperatures, 30 mw microwave source 50 mm 13 C 3 -L-alanine in glycerol-d l 8 / mm melittin (U- 13 C-Pro,Ala,Leu,Ile) D 2 O/H 2 O, 13 mm DOTOPA-Tempo, in 12 C-glycerol-d8/D2O/H20, 160 mm acetate, ph 3 10 mm DOTOPA-Tempo 6.8 khz MAS, 48 l sample volume 40 mm phosphate, p ph 7 T = 21 K 6.5 khz MAS, 68 l sample volume DNP enhancement = 30, DNP = 3.0 s T = 25 K DNP enhancement = 17, DNP = 8 s waves on waves on waves off waves off

40 (a) DNP-enhanced solid state NMR with MAS at very low temperatures, 30 mw microwave source 2D 13 C- 13 C spin diffusion (a) and fprfdr (b) spectra of U-PALI melittin. 2.5 hr each. CO ss P L L L L P P P L slice P slice 10 (b) A A A slice P, P 2, P slice C NMR frequency (ppm) ss P, P A Compared with earlier experiments at ~140 K (w/o DNP), ~3X less protein, ~70X less time. L P L L P P A A fre quency (p ppm) 13 C NMR f

41 extended interaction oscillator, 264 ± 0.5 GHz, ~700 mw CW (Communications & Power Industries, Inc.) Virginia Diodes transmitter system, 264 ± 10 GHz, 30 mw CW qausi-optical microwave transmission/polarization system "Martin-Puplett interferometer" (Thomas Keating, Ltd.)

42 Alexey Potapov EIO source ( 60) VDI source ( 15) no microwaves

43 THANK YOU! Wai-Ming Yau Wei Qiang Alexey Potapov Eric Moore Kent Thurber Marvin Bayro 43