Protein expression and purification. α-syn mutants containing a deletion of the C-terminus (amino acids 1-99), single cysteine mutants and single glutamic acid mutant were generated by using site-directed mutagenesis. Natural abundance and 15N-enriched α-syn and its variants were expressed in Escherichia coli BL21 (DE3) cells in either LB media or M9 minimal medium supplemented with 15NH4Cl. The cells were grown at 37 °C to an optical density at 600 nm (OD600) of 0.6 and induced with 1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) for 4 h. Cells were harvested by centrifugation at 6,300g for 10 min at 4 °C. FL-α-syn and FL-A90C were purified as described(79). For CT-α-syn and CT-A90C, cells were resuspended in 1 mM phenylmethanesulfonyl fluoride (PMSF), 50 mM MES, pH 6.0 and lysed in a high-pressure homogenizer (ATS Engineering). Cell lysates centrifuged at 48000g for 30 min at 4 °C. The supernatant was introduced into a SP cation exchange column (GE Healthcare) and the column eluted in a gradient of 0-1000 mM NaCl. Fractions containing the proteins were concentrated after SDS-PAGE analysis and then chromatographed on a Superdex 75 26/600 column (GE Healthcare) eluted with 50 mM MES and 200 mM NaCl, pH 6.0.
The human PDI gene was cloned into a pET-28a vector containing a His6-tag and a TEV protease cleavage site at the N terminus. The recombinant vector was transformed into E. coli BL21 (DE3). The cells were cultured in LB medium at 37 °C. When cell density reached an OD600 of 0.8, PDI expression was induced with 1 mM IPTG for 7 h. Cells harvested by centrifugation were resuspended in 20 mM Tris, 500 mM NaCl, pH 8.0 with freshly prepared 10mg/L protease inhibitors (Sigma-Aldrich). After lysis and centrifugation, the soluble fraction applied to a HisTrap column (GE Healthcare), which was eluted with a linear gradient of 0-500 mM imidazole. Protein was further purified by anion exchange chromatography with a Resource Q column (GE Healthcare) using 20 mM Tris, pH 8.0 and eluted by a linear gradient of 0-1000 mM NaCl, followed by size-exclusion chromatography with a Superdex 200 16/600 column (GE Healthcare) equilibrated with 20 mM Tris, 300 mM NaCl, pH 8.0. Protein concentration was determined either by absorbance of aromatic amino acids at 280 nm with a NanoDrop spectrophotometer (extinction coefficients is 5.96 and 1.49 for FL- and CT-α-syn, respectively), or by Bradford assay using BSA as a standard. Purified proteins were desalted using a HiTrap column (GE Healthcare), lyophilized and stored at -80 °C.
Liposome preparation. POPC, POPA and CL were purchased from Avanti Polar Lipids (Alabaster, AL) and used without purification. Liposomes containing 50% POPC and 50% POPA, and liposomes containing POPC and CL (mole ration of 9:1 for C1 liposomes and 7:3 for C3 liposomes) were prepared as described(79, 80).
Liposome co-flotation assay. Liposomes were incubated with FL- or CT-α-syn for 30 min at room temperature. An equal volume of iohexol [80% (w/v)] was added. The sample in 40% (w/v) iohexol was transferred to a thick-wall centrifugation tube (Beckman) before 300 μl of liposome buffer, 375 μl of 30% (w/v) iohexol and 375 μl of 35% (w/v) iohexol were added in that order without mixing. A SW60Ti rotor (Beckman) was used for gradient centrifugation at 28000g for 150 min at 4 °C. Lipid bound proteins were recovered from the 0% and 40% iohexol. The mixture was then deconstructed from the top into nine equal fractions prior to SDS-PAGE analysis. Image J software (National Institutes of Health) was used to quantify band intensity.
1H-15N Heteronuclear single quantum coherence (HSQC). Spectra from membrane-binding experiments were acquired using 0.1 mM 15N-enriched FL- and CT-α-syn dissolved in NMR buffer (20 mM HEPES, 100 mM KCl, pH7.0) plus 10% (v/v) D2O in the absence or presence of 12 mM liposomes. 1H-15N HSQC spectra from PDI binding experiments were acquired using 0.3 mM 15N-enriched FL- and CT-α-syn dissolved in 20 mM HEPES, 100 mM NaCl, 5 mM tris (2-carboxyethyl) phosphine (TCEP), pH 7.0, 10% (v/v) D2O in the absence, or presence of PDI at mole ratios from 10:1 to 1:1. Experiments were carried out at 15 °C on a Bruker Avance 800 or 850 MHz NMR spectrometer. Resonance assignments for α-syn are available from the BioMagResBank (entry number 16543). Data were analyzed with Sparky software. Intensity ratios were calculated by peak heights in the presence and absence of liposomes or PDI. CSPs of backbone amides were calculated according to Eq. 1(81), where ΔδH and ΔδN denote the chemical shift difference in the absence and presence of PDI in the 1H and 15N dimension, respectively.
1
KD was calculated using the fitting function shown in Eq. 2(82):
2
Where Δδobs is the observed FL- or CT-α-syn chemical shift minus the free FL- or CT-α-syn shifts, [P]t represents the concentration of FL- or CT-α-syn (300 mM), and [L]t represents PDI concentration, from 0 to 300 mM.
Paramagnetic relaxation enhancement (PRE). For spin label (MTSL) conjugation, single cysteine variants of 15N-enriched FL- and CT-α-syn (FL- and CT-A90C) were dissolved in NMR buffer and reduced with 5 mM DTT for 30 min. The proteins were then reacted with a 5-fold mole excess of MTSL at 4 °C for 16 h in the dark. Excess MTSL was removed by eluting samples over a HiTrap desalting column (GE Healthcare) into NMR buffer. Complete labeling was confirmed by LC-MS. Spin-labeled FL- and CT-α-syn were lyophilized or used directly. Intramolecular PRE experiments required 100 μM 15N-MTSL-labeled protein. PRE experiments were conducted in NMR buffer containing 10% (v/v) D2O at 15 °C using a Bruker Avance 850-MHz spectrometer. Diamagnetic samples were prepared by adding a 10-fold mole excess of ascorbic acid to the paramagnetic samples. PRE data were processed using NMRpipe-software and analyzed with Sparky- software. PRE effects were quantified as the ratios of peak intensities recorded in the paramagnetic state versus the diamagnetic state, respectively. The transverse relaxation rate enhancements (R2) were calculated with Eq. 3(83):
3
Where Idia, Ipara denote NMR signal intensity in the diamagnetic and paramagnetic state, respectively, the interval time Tb−Ta, was 17.2 ms for FL-α-syn and 20 ms for CT-α-syn.
Dynamic light scattering (DLS). FL- or CT-α-syn (20 μM) were incubated with 100 μM liposomes in a total volume of 1 ml of NMR buffer at room temperature for 30 min. Clustering activity was measured by DLS using a Protein Solutions DynaPro instrument (Malvern) in triplicate, with an average of 10 data points. Particle size distribution of liposomes for control experiments was monitored under same conditions.
Cyto c release. At least 107 SK-N-SH cells were used per experiment. The mitochondria were prepared with a Mitochondria Isolation Kit (Solarbio) for cultured cells. The procedure is described previously(84). Briefly, freshly isolated mitochondria were resuspended in ice cold buffer (10 mM HEPES, 2 mM K2HPO4, 10 mM succinate, 250 mM sucrose, 1 mM ATP, 0.08 mM ADP, 1 mM DTT). Aliquots were incubated with FL- and CT-α-syn at 37 °C for 1h. After incubation, the mixture was centrifuged at 13000g at 4 °C for 10 min and the supernatant interrogated using SDS-PAGE followed by WB with an anti-cyto c monoclonal antibody (Abcam).
Mitochondrial membrane potential. SK-N-SH cells were incubated with 50 μM FL- and CT-α-syn 37 °C for 5 h. The JC-1 kit (Sigma-Aldrich) was used according to manual. Images were acquired with a confocal fluorescence microscope (Leica TCS SP8). Fluorescence intensity was analyzed with Image J software.
Fibrillation monitored by thioflavin T (ThT) fluorescence. Lyophilized FL- and CT-α-syn were dissolved in 20 mM Tris, 100 mM NaCl, 2 mM TCEP, pH 7.5, 0.01% (w/v) NaN3 and filtered through a 0.22 μm Millex filter. A 1.5 mM ThT stock was prepared in same buffer. Samples (200 μl) containing either FL-/CT-α-syn or mixed with equivalent amount of PDI were dispensed along with a 2.5-mm glass bead in each well of a black, transparent bottomed, 96-well plate. The plate was sealed with a Al sealing tape (Corning) and incubated in a SpectroMax i3x microplate reader (Molecular Devices) at 37 °C with 500 s high-grade orbital shaking. ThT fluorescence was measured every 10 min using excitation and emission wavelengths of 444 nm and 482 nm, respectively. The data were analyzed using Eq. 4(11).
4
F(t) was normalized by dividing the largest value in each experiment, k represents the fibrillation rate constant and t1/2 is the aggregation half-time. The lag time tlag was calculated as: