Structural Characterization of Prion-like Conformational Changes of the Neuronal Isoform of Aplysia CPEB Bindu L. Raveendra, 1,5 Ansgar B. Siemer, 2,6 Sathyanarayanan V. Puthanveettil, 1,3,7 Wayne A. Hendrickson, 3,4 Eric R. Kandel, 1,3,4 Ann E. McDermott. 2 Supplementary Figures a b c d Supplementary Figure 1. Soluble form of Aplysia CPEB exists as oligomers in the native state. (a) Calibration curve for the superdex 200 column using 4 standards, (High Molecular weight Calibration Kit, GE, Cat No. 28-4038-42). The X-axis shows the log (molecular weight) and Y-axis is Kav (gel phase distribution coefficient). Kav = (Ve-V0)/(Vc-V0). Where Ve: elution volume, V0: void volume (43.08 ml) and Vc: total column volume (120 ml). (b) Estimation of apparent molecular weight of the
fractions (number 22, 31 and 32 from Fig. 2b and Supplementary Fig. 1c) as interpreted based on the molecular weight calibration curve. Two preparations of Aplysia CPEB (from Aplysia central nervous system and E.coli) have similar elution times and therefore should have a similar apparent molar mass. Monomer mass of endogenous Aplysia CPEB is 78.2 kda and recombinant protein is 83 kda. (c) Western analysis of native E.coli extract (native lysis) after fractionation by SEC. The different fractions obtained after SEC are numbered sequentially from 1-54. Aplysia CPEB in the soluble form in E.coli extract has similar pattern of molar mass distribution as of the Aplysia neuronal extract. Both of them showed mainly two populations: a small percentage of total population (< 20%) with a molecular weight corresponding to a dimer to tetramer (190-250 kda) and a larger percentage (>80%) as aggregated high molecular weight population (> 10-mer, >720 kda at fraction 22). Purified recombinant Aplysia CPEB in the soluble form also showed similar behavior in SEC and Western analysis. (d) Control experiment using kinesin heavy chain antibody. Western analysis of native CNS extract of Aplysia after fractionation by SEC. The protein kinesin heavy chain is analyzed in a similar way as for CPEB. The data for kinesin heavy chain showed that in the soluble form it exists mainly in two populations. The first fraction has an apparent molar mass corresponding to a dimer and the second fraction has very high apparent molecular weight indicating that kinesin might also exists as a homo oligomer or a high molecular weight hetero oligomeric protein complex. Kinesin heavy chain known to exists as dimer and high molecular weight protein complexes, which is in agreement with the data we obtained from this experiment.
Supplementary Figure 2: Analysis of purified soluble Aplysia CPEB by size exclusion chromatography light scattering (SEC-LS). (a) Purified recombinant full length Aplysia CPEB in PBS with 2 M urea was analyzed by size exclusion chromatography followed by light scattering. SEC is used as a fractionation column. The solid line indicates the trace from the UV detector and dotted line is the signal from LS detector at 90º. The dots are the weight average molecular weight for each slice measured every second. The molecular weight is calculated from light scattering signal and is shown on the right side Y-axis. The oligomeric state has an average radius of gyration (Rg) around 25 nm. (b) The hydrodynamic radius (Rh) distribution of the purified Aplysia CPEB full length protein in PBS in the presence of 2 M urea. The dotted line shows the Rh distribution derived from the light scattering signal. The right side of the Y-axis is the molecular weight of the oligomer calculated from light scattering experiment. The Rh value for the oligomeric fraction of Aplysia CPEB is found similar to the observed radius of gyration (Rg), around 25 nm, suggesting a hollow spherical shape for these oligomers. (c) Comparison of SEC-LS analysis of purified recombinant full length Aplysia CPEB in PBS buffer with 2 M urea (blue) and 4 M urea (red). The solid line indicates the trace from the UV detector and dotted line from LS detector at 90 degree. Purified full length Aplysia CPEB in PBS with 4 M urea eluted at a lower elution volume (5 6.5 ml) in the SEC indicating a high apparent molecular weight. But from the light scattering data, this fraction has an average molecular weight of ~80 200 kda equal to the mass of the monomer to the trimer. Despite the fraction showed a very low elution volume in SEC (5 6.5 ml) compared to the protein in PBS with 2 M
urea buffer, it is having a lower molecular weight by light scattering data. The larger size observed for this molecules might be due to the extended conformation resulting from the partial denaturing of the protein under this condition.
Supplementary Figure 3. Solid-state NMR of fiber form of full length Aplysia CPEB and isolated prion domain. (a) 13 C- 13 C DARR spectrum of full-length Aplysia CPEB recorded at a 1 H frequency of 900 MHz, 20 khz MAS, and mixing time of 50 ms. The assignment of some of the amino acid types is indicated. The average Cα-Cβ cross peak positions corresponding to an α-helical (red), extended β-sheet (orange), and random coil (blue) conformation for the amino acids Thr, Ser, Gln, and Ala, as well as the corresponding Cα-CO cross peaks for Thr and Gln are marked with ellipsoids. The average chemical shifts as well as the standard deviation of these shifts (corresponding to the width of the ellipsoids) were taken from Wang & Jardezky. 48 The full 13 C connectivity of Gln in α-helical and β-sheet conformation is shown with red and green bars, respectively. As illustrated in Fig. 7, this spectrum is very similar to the 2D DARR spectrum of the isolated prion domain of Aplysia CPEB. (b) Assignment of Gln 13 C shifts. A detail of the 13 C- 13 C DARR spectrum of Figure 7a is shown in red (CP DARR). The equivalent spectrum recorded with a direct 13 C excitation instead of 1 H- 13 C CP to create the initial polarization is shown in blue (DE DARR). The contours were adjusted in a way that only the most intense cross peaks (i.e. the cross peaks of Gln, the most abundant amino acid of the PRD) are visible. Both spectra show the entire 13 C spin system of Gln. However, the CP DARR (red) gave a higher intensity for the Gln in a β-sheet conformation (solid-lines) and the DE DARR (blue) for Gln in a random-coil and α-helical conformation (dashed lines). The figure illustrates that the high abundance of Gln (~40%) in the PRD and the very intense cross peaks resulting from that make it is possible to unambiguously assign the Gln 13 C shifts in our 2D DARR spectra.
d 75 50 30 25 20 17 e 28 KDa! PRD 10min 30min! 7 KDa! 15 f Supplementary Figure 4: Proteolysis experiments on Aplysia CPEB ( a) Mass spectrometric data of the trypsin digested full length Aplysia CPEB fibers. The red color shows the sequence of the fragment obtained by mass spec analysis after digestion of full length Aplysia CPEB fibers by trypsin. (b) Mass spectrometric analysis of full length Aplysia CPEB in the soluble form. The red segments show the sequence of the fragments obtained by mass spec analysis after trypsin digestion. (c) Theoretical prediction of trypsin digestion sites for full length Aplysia CPEB using the program Expassy Peptide cutter. The trypsin cleavable sites are shown in red colored letters. (d) Coomassie stained PAGE gel showing a 20 kda trypsin resistant fragment obtained from full length Aplysia CPEB fibers after digestion. The presence high molecular weight bands (>20 KDa) in the gel indicates that the fragment might be very prone to aggregation resulting in high molecular weight oligomers. (e) Proteolysis (proteinase K) of the isolated prion domain (PRD) and full length Aplysia CPEB fibers prepared from purified recombinant protein. A 7 kda protease resistant fragment is present in both samples after the proteolysis. The presence of the same band from both full length Aplysia CPEB and PRD indicates that the fragment comes from the PRD of the protein. The identification of band by Mass spectrometry and N-terminal sequencing was unsuccessful. (f) Amino acid sequence of a protease- fragment cleaved from the full length Aplysia CPEB fibers. This sequence has four potential resistant trypsin cleavage sites (red) that are not cut by the enzyme when Aplysia CPEB is in the fibrillar form. This fragment is part of the PRD of the neuronal isoform of Aplysia CPEB and, therefore, not present in
the developmental isoform. In contrast, the soluble form of full length Aplysia CPEB does not exhibit such pronounced protease resistance.
Supplementary Figure 5: Comparison of combined MS scans from soluble and insoluble forms of Aplysia CPEB after Trypsin digestion. Scans across the identical time range for both analyses were combined using the combine function of the MassLynx 4.1 software and compared. (a,b) Combined scans from soluble and insoluble forms, respectively, showing the 3+ ion of peptide 121-143 (EQLQQQQLQLQQQLQQQLQHIQK) present in the soluble form only. (c,d) Combined scans from the soluble and insoluble forms, respectively, showing the 3+ion of peptide 112-143 (QQLQQQQQQEQLQQQQLQLQQQLQQQLQHIQK) present in the soluble form only. (e,f) combined scans from the soluble and insoluble forms, respectively, showing the 2+ion of peptide 144-171 (EPSSHTYTPGSPELQSVLNYANVPLSK) present in the soluble form only.
Supplementary Table 1 (NMR): Comparison of the chemical shifts extracted from the 1 H- 13 C HETCOR spectrum shown in Fig. 5c to the average chemical shifts of α-helical, β-sheet, and random coil secondary structure elements reported by Wang & Jardetzky 1 (the standard deviation is given in parenthesis). This analysis shows that the chemical shifts of the dynamic parts detected in Aplysia CPEB fibers are in agreement with random-coil values or, in some cases, α-helical values. 1. Wang, Y. & Jardetzky, O. Probability-based protein secondary structure identification using combined NMR chemical-shift data. Protein science : a publication of the Protein Society 11, 852-861, (2002).