Structural insights into an equilibrium folding intermediate of an archaeal ankyrin repeat protein

Löw et al. 10.1073/pnas.0710657105.

Supporting Information

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SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Table 1
SI Table 2
SI Materials and Methods

SI Figure 7

Fig. 7. Sequence alignment of tANK from Thermoplasma volcanium with human CDK inhibitor p19^INK4d. Identical and similar residues are color coded in red. Helices of individual ARs above L36 are indicated.

SI Figure 8

Fig. 8. Analytical ultracentrifugation equilibrium sedimentation profile of tANK at 1.95 M GdmCl fits to a single exponential model with M_r = 22,700 ± 1,600, indicating that the intermediate state exists as a monomer in solution. The measurement was performed at 20°C, 1 mg/ml and 20,000 rpm (32,170 ´ g) for 62 h. Residual deviation of the fit from the experimental data are shown at the bottom.

SI Figure 9

Fig. 9. 2D ¹⁵N-TROSY-HSQC spectrum of 2.0 mM ²H/¹⁵N tANK at 25°C in 90%/10% H₂O/²H₂O, pH 7.4. The assigned cross-peaks of the amide backbone are labeled using the one-letter amino acid code and the sequence position. Boxes indicate resonance signals that show cross-peak intensities below the plotted contour level.

SI Figure 10

Fig. 10. Temperature dependence of the slowest two apparent rate constants for refolding of tANK. Refolding conditions were 0.9 M GdmCl, pH 7.4. The activation energy (E_A) and the preexponential factor A for both reactions were obtained using the Arrhenius equation (solid line). The slowest refolding reaction (open triangles) shows a temperature-independent activation energy of 78.5 3 kJ mol^-1 (A = 10^12.2 s^-1). The activation parameter for the faster folding reaction (open circles) result in 47.5 3 kJ mol^-1 (A = 10^9.17 s^-1).

SI Figure 11

Fig. 11. Fibrillation of tANK. 1 mM tANK was incubated at (A) 2 M GdmCl and (B) 0 M GdmCl. Aliquots were transferred at different time points to 2 M GdmCl solutions containing ANS, incubated for 5 min, and fluorescence spectra were recorded. Preincubated protein at 2 M GdmCl strongly binds ANS (violet line), whereas freshly dissolved protein at 2 M GdmCl does not (black line in A and B), although the intermediate is maximally populated. (C) Fibrillation recorded by Thioflavin T fluorescence. Kinetics show a lag phase typical for fibril formation. Fibrillation speed strongly depends on concentration (data not shown), temperature, and the percentage of populated intermediate. (D) Electron microscopy of negatively stained tANK aggregates. Fibrils were formed by incubation of 1 mM protein in 20 mM Na-phosphate, 2 M GdmCl, pH 7.4 at 25°C for 2 h. (Scale bar, 200 nm.)

SI Materials and Methods

X-Ray Diffraction and Structural Refinement. A redundant dataset from a single iodine derivatized crystal was collected in house at -180°C with Cu Ka radiation (l = 1.5418 Å) using a rotating-anode source (RA Micro 007, RigakuMSC) and image plate detector (R-AXIS IV++, RigakuMSC). The crystal diffracted to a resolution of 1.83 Å. Data were indexed and scaled in Mosflm and Scala programs respectively (1-3). The anomalous scattering signal from iodine atoms was used for substructure determination using Single wavelength Anomalous Diffraction (SAD). Eleven iodine positions with occupancy between 1 and 0.05 were determined with the program SHELXD (4) using diffraction data up to 2.3 Å. For phase calculation and further density modification using the program SHELXE, the number of iodine atoms was truncated to 7 and only those with occupancy greater than 0.2 were used. At this stage, the space group could be determined as P6₄22.

The resulting electron density had excellent quality and was used for automatic main-chain tracing and side-chain docking carried out with the ARP/wARP software (5) and the CCP4 suite (3). The model from the autobuild process comprised two chains (residues from 20 to 188). The missing residues were rebuilt and the structure manually verified against sigma weighted difference Fourier maps using Coot (6) program. Refinement against a maximum likelihood target with a combination of restrained and TLS refinement was performed using Refmac (7). During refinement, 5% of the reflections were randomly chosen and left out for cross-validation using the free R factor. The structure has been refined to an R factor of 18.6% and R_free of 20.22% respectively (SI Table 1).

A dataset from a single non derivatized crystal was first collected in house (resolution 1.75 Å) and later at BESSY to the resolution of 1.65 Å. The synchrotron data were collected at beam line BL 14.1 equipped with a fast scanning 225 mm CCD-mosaic detector from MARRESEACH (Norderstedt, Germany) at a wavelength of 0.9123 A. Graphical analysis and refinement was carried out as described for the iodine derivatized crystal. The final model consists of residues 6 to 189, the missing N-terminal amino acids show a non interpretable electron density map and are most likely disordered. There are 497 solvent molecules included in the model, as well as 8 sulfate ions, 3 Cl ions, 4 1,3-butanediol molecules, 6 glycerol molecules and 1 Tris molecule. For some residues the electron density indicated multiple side chain conformations in the crystal. The model was refined to an R factor of 15.58% and R_free of 17.83%. The refined model has good geometry as judged by PROCHECK (3) - Ramachandran statistics showed 90.4% of amino acids in favoured regions, 9.0% in allowed regions, 0.6% generously allowed and none in disallowed regions.

ANS and Thioflavin T Fluorescence. To study ANS binding properties, protein was incubated in 20 mM Na-phosphate, 2 M GdmCl at a concentration of 1 mM at 25°C for fibril formation to occur. A 1 mM sample without GdmCl was used as a reference. To determine ANS fluorescence, samples were briefly mixed and then diluted to a final concentration of 5 mM with 50 mM ANS in 20 mM Na-phosphate, 2 M GdmCl, pH 7.4. Fluorescence spectra were recorded at an emission wavelength of 410 to 600 nm upon excitation at 370 nm at 25°C. Fibril formation was also monitored by Thioflavin T fluorescence according to standard procedures with minor modifications (8). Optimization of protein concentration, temperature and amount of GdmCl was necessary to avoid fibril formation for later NMR experiments.

Electron Microscopy (EM). For EM analysis, carbonized copper grids (Plano, Wetzlar, Germany) were pretreated for 1 min with bacitracin (0.1 mg/ml). After air drying, protein (preincubated in 2 M GdmCl) that had been diluted with 20 mM Na-phosphate to final concentrations of 0.5 mg/ml was applied for 3 min. Subsequently, grids were again air dried. Protein (fibrils) was negatively stained with 1% (wt/vol) uranyl-acetate and visualized in a Zeiss EM 900 electron microscope operating at 80 kV.

Limited Proteolysis and Mass Spectrometry. tANK (3 mg/ml) was incubated with 10% Chymotrypsin at 15°C in 20 mM Na-phosphate. The reaction was stopped at certain time by addition of sample buffer and subsequent boiling. Fragments were separated on a 4-20% gradient SDS gel resulting in 2 distinct fragments with an estimated mass of 18.5 kDa and 10 kDa (data not shown). Stained protein bands were excised, washed and digested with modified trypsin. Peptides were applied to MALDI target plates as described (9). Mass spectra were obtained automatically by MALDI-TOF MS in reflection modes (Voyager-DE-STR), followed by automatic internal calibration using tryptic peptides from autodigestion. The spectra were analyzed using the software MoverZ from Genomic Solutions (http://bioinformatics.genomicsolutions.com/MoverZDL.html) with a signal to noise ratio threshold of 3.0. Resulting peptide mass lists were used to match the thermophilc AR sequence using the Mascot Search engine (www.matrixscience.com).

Ultracentrifugation Measurements. Sedimentation analyses were conducted using an analytical ultracentrifuge Optima X-LA (Beckman Instruments, Palo Alto, CA) equipped with two channel cells and an An50 Ti rotor. For these analyses, the protein concentration was varied between 0.1 and 1 mg/ml. Dilutions were performed with 20 mM Na-phosphate, pH 7.4, 1.95 M GdmCl. Native protein was analyzed in the same buffer in the absence of GdmCl. The samples were measured at 20°C and a wavelength of 280 nm. Sedimentation velocity was carried out at 40,000 rpm, scans were taken every 10 min. Sedimentation equilibrium was performed at 20,000 rpm. The data were analyzed with the software provided by Beckman Instruments. Viscosity and density of the buffer containing 1.95 M GdmCl were calculated as described (10).

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