Supporting Information

Intermolecular Alignment in Y145Stop Human Prion Protein Amyloid Fibrils Probed by Solid-State Nuclear Magnetic Resonance Spectroscopy
Jonathan J. Helmus, Krystyna Surewicz, Marcin I. Apostol, Witold K. Surewicz, and Christopher P. Jaroniec,*

Department of Chemistry, The Ohio State University, Columbus, Ohio 43210 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
E-mail: jaroniec@chemistry.ohio-state.edu

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Preparation of huPrP23-144 Amyloid Fibrils Isotopically enriched and natural abundance huPrP23-144 was overexpressed in Escherichia coli BL21 Star (DE3) One Shot cells (Invitrogen) and purified as described previously.1 The
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C and/or
15

N labeled proteins were prepared using minimal medium containing the following additives,2-4

N-

huPrP23-144: 1 g/L 15NH4Cl, 4 g/L glucose; 1,3-13C-huPrP23-144: 1 g/L 14NH4Cl, 2 g/L NaH12CO3, 2 g/L [1,3-13C]glycerol; 2-13C-huPrP23-144: 1 g/L 14NH4Cl, 2 g/L NaH13CO3, 2 g/L [2-13C]glycerol; 1,313

C,15N-huPrP23-144: 1 g/L

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NH4Cl, 2 g/L NaH12CO3, 2 g/L [1,3-13C]glycerol; 2-13C,15N-huPrP23-

144: 1 g/L 15NH4Cl, 2 g/L NaH13CO3, 2 g/L [2-13C]glycerol. All 13C and 15N-enriched compounds were purchased from Cambridge Isotope Laboratories (Andover, MA). Amyloid fibrils were formed at 25 oC without agitation for ~24 h by dissolving lyophilized huPrP23-144 in ultrapure water at a concentration of 5 mg/ml, followed by addition of pH 6.4 potassium phosphate buffer to a final concentration of 50 mM. Fibrils were characterized by atomic force microscopy to confirm sample homogeneity, pelleted by low-speed centrifugation, washed with several aliquots of 50 mM pH 6.4 potassium phosphate buffer, and centrifuged into Varian 3.2 mm limitedspeed zirconia rotors for solid-state NMR analysis. The rotors contained ~15 mg of total protein and were sealed using custom-made spacers (Revolution NMR, Fort Collins, CO) to prevent sample dehydration during NMR experiments. A total of six samples were used in this study, corresponding to huPrP23-144 fibrils formed from: (1) 1,3-13C,15N-huPrP23-144, (2) a physical mixture of 1,3-13C,15NhuPrP23-144 and natural abundance huPrP23-144 in a 1:3 molar ratio, (3) a physical mixture of 1,3-13ChuPrP23-144 and
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N-huPrP23-144 in a 1:1 molar ratio, (4) 2-13C,15N-huPrP23-144, (5) a physical

mixture of 2-13C,15N-huPrP23-144 and natural abundance huPrP23-144 in a 1:3 molar ratio, and (6) a physical mixture of 2-13C-huPrP23-144 and 15N-huPrP23-144 in a 1:1 molar ratio. Solid-State NMR Spectroscopy NMR experiments were carried out using a three-channel Varian spectrometer operating at the frequencies of 499.8 MHz for 1H, 125.7 MHz for 13C, and 50.6 MHz for 15N, and equipped with 3.2 mm triple-resonance T3 and BioMAS5 probes in 1H-13C-15N configuration. All experiments employed MAS rates of 11.111 kHz, actively regulated to ca. 3 Hz using a Varian MAS control unit. Effective sample temperatures during the experiments were maintained at ~5 oC using a variable temperature stack and temperature control unit. 2D
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N-13C chemical shift correlation spectra for the huPrP23-144 fibril samples were acquired

using the z-filtered TEDOR pulse scheme,6 with t1,max(15N) 14 ms, t2,max(13C) 24 ms, 27.8 kHz 15N recoupling pulses, 5.4 ms z-filter periods with simultaneous application of ~50 kHz continuous-wave 1H S2

field to suppress undesirable antiphase and multiple-quantum coherences, and 100 kHz and 70 kHz TPPM 1H decoupling7 during the TEDOR mixing and chemical shift evolution/acquisition periods, respectively. The TEDOR mixing times, TEDOR, employed ranged from 1.44 ms to 18.0 ms. 1D TEDOR trajectories that report simultaneously on all intermolecular
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N-13CO and

15

N-13C

distances in huPrP23-144 fibrils were recorded for the mixed 1,3-13C/15N and 2-13C/15N samples, respectively. For the 15N-13CO trajectory, a series of 30 13C 1D spectra were collected with TEDOR varied between 0.72 ms and 21.6 ms in 0.72 ms incrementsthis set of experiments also employed a bandselective 360 s rSNOB8 13C refocusing pulse centered in the carbonyl region to suppress any residual
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CO-13C J-couplings.6 For the

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N-13C trajectory, a series of 12

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C spectra were collected with

TEDOR varied between 1.8 ms and 21.6 ms in 1.8 ms increments. Estimation of the -Strand Spacing in huPrP23-144 Fibrils from Solid-State NMR Data As discussed in the main text, the multiple intermolecular correlations observed in 2D
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N-13C

TEDOR correlation spectra for the mixed 1,3-13C/15N and 2-13C/15N huPrP23-144 fibril samples (c.f., Figures 1 and S1) are consistent with a parallel in-register alignment of the -strand segments within the amyloid core. The -strand spacing was estimated by recording 1D TEDOR trajectoriesthat report on all intermolecular
13 15

N-13CO and

15

N-13C distances, respectivelyfor the mixed 1,3-13C/15N and 2-

C/15N fibril samples as described above, and comparing these experimental trajectories with ones The simulated 1D TEDOR trajectories were generated using the SPINEVOLUTION program9 as

calculated for model spin systems (c.f., Figure 2). follows. First, a series of atomic coordinate files corresponding to model spin systems to be used by SPINEVOLUTION for the nuclear spin dynamics simulations was built based on the X-ray structure of amyloid-like microcrystals of the peptide GNNQQNY10 displaying a parallel in-register alignment of the -strands (PDB ID 2OMM), where the spacing between adjacent strands, d, (c.f., Figure 2) was varied between 3.5 and 6.0 in 0.1 increments. Next, for each -strand spacing SPINEVOLUTION was used to calculate three different TEDOR buildup trajectories for a to
15 15 13

CO or

13

C spin on the central

strand (denoted as 0 in the structural models shown in Figure 2) arising from dipole-dipole couplings N nuclei located within 6 of the
13

CO/13C spin on the adjacent +1 and -1 strands

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specifically, the three trajectories corresponded to the 13C nucleus coupled to: (1) two nearest-neighbor N spins on strand +1, (2) two nearest-neighbor N spins on strand -1, and (3) four nearestneighbor 15N spins on strands +1 and -1, indicated by spheres in the models in Figure 2. This was followed by averaging the three calculated trajectories in a 1:1:1 ratio, in accord with the 13C-15N spin topologies that are expected for amyloid fibrils grown from an isotropic 1:1 physical mixture of 13C and S3

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N-labeled protein monomers. Finally, the dependence of the data analysis on additional parameters not
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related to the -strand spacing, including nuclear spin relaxation and overall spectral intensity, was eliminated as follows. First, the CO/13C intensities for the experimental TEDOR trajectories were
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scaled by the corresponding intensities in 1D cross-polarization

C reference spectra recorded under

identical conditions. Second, the transverse 13C spin relaxation rates were determined by standard spinecho methods (under the experimental conditions of this study the collective 13CO and 13C relaxation rate constants for the mixed 1,3-13C/15N and 2-13C/15N huPrP23-144 fibril samples were found to be 82 s-1 and 136 s-1, respectively) and subsequently used to scale the calculated TEDOR trajectories by a factor of exp{R2 TEDOR}, where R2 is the 13CO/13C transverse relaxation rate. Note that the effects of 13C-13C J-couplings on the spin dynamics are negligible for these TEDOR experiments due to the use of the sparse
13

C labeling scheme, which eliminates most one-bond C pulse for the

15

13

C-13C J-couplings,3,4 and/or the

band-selective

13

N-13CO measurements. Thus, in summary simulations of the 1D

TEDOR trajectories contained no adjustable parameters other than the -strand spacing, d. For each value of d in the grid the root-mean-square deviation (RMSD) was determined between the experimental and simulated trajectories; d = 4.4 was found to give the lowest RMSD values for both the 15N-13CO and 15N-13C data sets (c.f., Figure 2). X-ray Fiber Diffraction Fibers were pelleted using low speed centrifugation, washed with water to eliminate salts, and repelleted. The pellet was subsequently resuspended with a small volume of water. The resuspened pellet was pipetted into a space between the ends of two glass rods. As the pellet dried, fibers aligned themselves between the rods. Diffraction images were collected at the Cleveland Center of Membrane and Structural Biology (CCMSB) using a Rigaku MicroMax-007HF copper anode X-ray source and a Rigaku Saturn 944+ CCD detector.

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Figure S1. Regions of 15N-13CX (NCX) TEDOR correlation spectra corresponding to side-chain methyl 13 C for huPrP23-144 fibrils. The spectra were recorded at 500 MHz 1H frequency, 11.111 kHz MAS rate and effective sample temperature of ~5 oC using the z-filtered TEDOR pulse scheme with 15N-13C dipolar mixing times, TEDOR, indicated in the spectra for amyloid fibril samples prepared from (A) 1,313 15 C, N-huPrP23-144, (B) a mixture of 1,3-13C,15N-huPrP23-144 and natural abundance huPrP23-144 in a 1:3 molar ratio, and (C) a mixture of 1,3-13C-huPrP23-144 and 15N-huPrP23-144 in a 1:1 molar ratio. The experiment times for spectra in panels (A-C) were ~66, ~108 and ~212 h, respectively. The intraresidue 15N-13C correlations that are relevant to the current study are labeled in black font. Inter-residue correlations are labeled in red font.

Figure S2. Typical nearest-neighbor and next-nearest-neighbor intermolecular (A) 15N-13CO and (B) 15 N-13C distances (in ) for amyloid-like microcrystals of the model peptide GNNQQNY displaying a parallel in-register alignment of the -strands (PDB ID 2OMM).10 All other intermolecular 15N-13C distances were > 6 . Side-chain atoms are not shown for clarity.

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Figure S3. X-ray diffraction of aligned huPrP23-144 fibrils. (A) The X-ray diffraction image shows a pattern with a prominent 4.65 reflection along the meridional or fiber axis corresponding to the spacing between -strands along this axis. There are also five weak equatorial reflections at 5.2, 6.2, 8.1, 9.5 and 12 arising from the spacing between stacks of -sheets within the fiber structure. (B) A graphical comparison of the intensity of the reflections along the meridian (red) and equator (blue) of the fiber diffraction pattern with peaks labeled above in units of .

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References Helmus, J. J.; Surewicz, K.; Nadaud, P. S.; Surewicz, W. K.; Jaroniec, C. P. Proc. Natl. Acad. Sci. USA 2008, 105, 6284-6289. (2) Cai, M. L.; Huang, Y.; Sakaguchi, K.; Clore, G. M.; Gronenborn, A. M.; Craigie, R. J. Biomol. NMR 1998, 11, 97-102. (3) LeMaster, D. M.; Kushlan, D. M. J. Am. Chem. Soc. 1996, 118, 9255-9264. (4) Castellani, F.; van Rossum, B.; Diehl, A.; Schubert, M.; Rehbein, K.; Oschkinat, H. Nature 2002, 420, 98-102. (5) Stringer, J. A.; Bronnimann, C. E.; Mullen, C. G.; Zhou, D. H. H.; Stellfox, S. A.; Li, Y.; Williams, E. H.; Rienstra, C. M. J. Magn. Reson. 2005, 173, 40-48. (6) Jaroniec, C. P.; Filip, C.; Griffin, R. G. J. Am. Chem. Soc. 2002, 124, 10728-10742. (7) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. J. Chem. Phys. 1995, 103, 6951-6957. (8) Kupce, E.; Boyd, J.; Campbell, I. D. J. Magn. Reson. B 1995, 106, 300-303. (9) Veshtort, M.; Griffin, R. G. J. Magn. Reson. 2006, 178, 248-282. (10) Sawaya, M. R.; Sambashivan, S.; Nelson, R.; Ivanova, M. I.; Sievers, S. A.; Apostol, M. I.; Thompson, M. J.; Balbirnie, M.; Wiltzius, J. J.; McFarlane, H. T.; Madsen, A. .; Riekel, C.; Eisenberg, D. Nature 2007, 447, 453-457. (1)

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