SUPPLEMENTAL TABLE LEGENDS

Transcription

1 SUPPLEMENTAL DATA Recombinant protein details The DNA constructs used for this study add a His-tag (6 His) and an 8 residue spacer between the His-tag and the first residue (Asp) of mature human apoa-i sequence. The spacer contains a thrombin and an acid cleavage sites. These recombinant apoa-is have been extensively used and characterized by our and other laboratories with His-tag and after His-tag removal by acidcleavage. No significant changes in protein structure or function have been detected in the presence of the additional residues at the N-terminal (1-5). Furthermore, for all the biophysical, biochemical and cellular experiments reported in this study, no significant differences were detected between His-tag-containing wild type recombinant apoa-i (WT-apoA-I) and plasma purified apoa-i. Thermal treatment protocols - Thermal treatments were performed by equilibrating 100 μl of apoa-i stock solutions to room temperature for 30 min, followed by incubation at the indicated temperature for 1 h and re-equilibration to room temperature for min. After centrifugation at low speed (<1000 rpm), protein concentration of supernatant was measured by BCA. Thermal treatment-dependent protein loss by precipitation was < 5% for all tested temperatures. For sample analysis by electrophoresis, chromatography, spectroscopy and for cell treatments, samples were rapidly diluted to the required concentration just after thermal treatment and re-equilibration to room-temperature. Evaluation of the apparent mass of self-associated species by Size Exclusion Chromatography (SEC) - In SEC experiments, the apparent mass of protein samples was determined by comparing their distribution coefficients (K av ) with those of globular proteins of known mass from Gel Filtration Calibration Kit (GE Healthcare) and Gel Filtration Standard (Bio-Rad): bovine thyroid thyroglobulin, 669 KD; horse spleen ferritin, 440 KD; bovine liver catalase, 232 KD; bovine heart lactate dehydrogenase, 140 KD; bovine serum albumin, 67 KD; chicken ovalbumin, 44 KD; horse myoglobin, 17 KD; vitamin B12, 1,350 D. K av values were calculated as: (V e V 0 )/(V t V 0 ). Where V 0 is the void volume, V t is the total interstitial volume, and V e is the elution volume. Standard proteins K av values were plotted against log(mass). The points were fitted by linear regression analysis and unknown sample masses were calculated by interpolation (2,6). Apparent masses of self-associated species were within experimental errors when calculated by Superose 6 or Superdex 200 column calibrations. Dynamic Light Scattering (DLS) - The molecular weight of protein species in solution was measured by DLS on a DynaPro-MSXTC DLS instrument. Samples (40-80 μl) were manually injected and illuminated by a 25 mw, 750 nm wavelength, solid state laser. Scattered light was collected at 90. DLS measurements of ΔW[High]60C were performed at 25 C and over the mg/ml concentration range. Solutions of BSA and chymotrypsinogen (Sigma Chemical Co.) in double-distilled water were used as reference standards. The data were analyzed with the Dynamics V6 software package. Preparation of lipid-free W-apoA-I self-association subclasses - Hexadecamers ( W[High] SEC F1) and octamers ( W[High] SEC F3) of W-apoA-I were isolated from W[High] by SEC on a Superdex 200 prep grade column with elution as described in Experimental Procedures and supplemental Fig. S1. Tetrameric ( W[High]60C) and predominantly monomeric ( W[High]90C) W-apoA-I were generated by incubating 100 μl of W[High] at 60 or 90 C, respectively, for 1 h (Fig. 5A & 8). To verify the viability of these samples as models for testing the effect of apoa-i self-association on cellular lipid release, the stability of the self-associated subclasses was measured in conditions similar to those of cell culture experiments. By NDGGE (Fig. 8) and SEC (data not shown) analysis non-significant disruption or interconversion between subclasses were observed in hexadecameric and octameric samples stored at 4 C for few months (data not shown) or incubated at 37 C for 24h (Fig. 8). Tetrameric and monomeric samples remodeled towards formation of high-order self-associated species if stored at 4 C for as short as 24 h (data not shown). But minimal interconversion between subclasses was observed for 1

2 24h incubation at 37 C (Fig. 8). However, only high concentration samples ( 0.5 mg/ml) could be assessed by these techniques. To indirectly survey the potential disruption of W-apoA-I high-order selfassociated species at temperature and concentrations typical for cell culture experiments (2-20 μg/ml), W[High] was diluted to 10 μg/ml, incubated at 37 C, and its ellipticity was recorded for up to 25h (supplemental Fig. S2). A reduction of only about 2% of the high α-helical content (> 80%) of W[High] suggests that hexadecamers and octamers were non-significantly disrupted at these incubation conditions. Pressure Perturbation Calorimetry (PPC) - PPC data were recorded with a VP-DSC equipped with PPC accessory as described in (7). A nitrogen gas pressure of 5 bar was applied to the cell with a programmed regime in the context of a DSC experiment. The protein samples were prepared as described for DSC (0.5 mg/ml). The heat effects associated with such pressure changes were used to determine volume expansion coefficient of the solute on an absolute scale, α v (T). Buffer-buffer, buffer-water and water-water baselines used for data analysis were fitted by fourth-order polynomial with an apoa-i partial specific volume of 0.88 cm 3 g -1 (7). Data were recorded in mid-gain low-noise mode. SUPPLEMENTAL TABLE LEGENDS Tab. S1. DLS analysis of ΔW[High]60C. Means and SD values from four independent experiments are reported. After heating, ΔW[High]60C was diluted to the reported concentrations and analyzed by DLS. For all concentrations, the mass of the self-associated species is consistent with the predicted mass of ΔW-apoA-I tetramers, within the experimental error. Thus, dilution up to 0.1 mg/ml does not affect the self-association state of ΔW[High]60C. SUPPLEMENTAL FIGURE LEGENDS Fig. S1. SEC isolation of self-associated ΔW-apoA-I subclasses using a Superdex 200 prep grade column. Representative chromatogram (Panel A) and NDGGE analysis (3 μg protein/lane, panel B) of collected fractions. Injection of ~2 mg of ΔW[high] (lane 2) yielded ~0.3 mg of hexadecamers (F1, lane 3) and ~0.5 mg octamers (F3, lane 4), corresponding to ~ 40% of combined final yield. By NDGGE, isolated samples purity was > 90%. Molecular weight markers (High Molecular Weight Calibration Kit from GE Healthcare). WT[high] is shown in lane 1. Fig. S2. Far-UV CD spectroscopic analysis of WT[High] and ΔW[High] at concentration and temperature conditions similar to those used in cell culture experiments. Stock solutions (~3.0 mg/ml) were diluted to 10 μg/ml in PB and incubated at 37 C for up to 25h. Fig. S3. PPC of WT[High] (black) and ΔW[High] (light gray) at 0.5 mg/ml in PB. Fig. S4. Edmundson helical wheel representation of amino acids surrounding the three Trps predicted to be in α-helical regions (W50, W72, and W108). Helical projections were generated as an idealized α18/5 helix for residues (panel A) and α11/3 helices for residues (panel B) and (panel C) (8). The color code is dark grey for hydrophobic, white for polar and uncharged, and pink for charged residues. Trps are highlighted in red. Prediction of the orientation of the amphipathic wheels was solely based on clustering of hydrophobic residue on a sector of the wheel. The apolar and polar solvation space is represented by yellow and blue backgrounds, respectively. 2

3 SUPPLEMENTAL REFERENCES 1. Cavigiolio, G., Geier, E. G., Shao, B., Heinecke, J. W., and Oda, M. N. (2010) J Biol Chem 285(24), Cavigiolio, G., Shao, B., Geier, E. G., Ren, G., Heinecke, J. W., and Oda, M. N. (2008) Biochemistry 47(16), Martin, D. D., Budamagunta, M. S., Ryan, R. O., Voss, J. C., and Oda, M. N. (2006) J Biol Chem 281(29), Oda, M. N., Forte, T. M., Ryan, R. O., and Voss, J. C. (2003) Nat Struct Biol 10(6), Shao, B., Bergt, C., Fu, X., Green, P., Voss, J. C., Oda, M. N., Oram, J. F., and Heinecke, J. W. (2005) J Biol Chem 280(7), Nanjee, M. N., and Brinton, E. A. (2000) Clin Chem 46(2), Benjwal, S., and Gursky, O. (2010) Proteins 78(5), Segrest, J. P., Jones, M. K., Mishra, V. K., Anantharamaiah, G. M., and Sidney A. Simon, T. J. M. (2002) Experimental and computational studies of the interactions of amphipathic peptides with lipid surfaces. In. Peptide-Lipid Interactions, Academic Press 3

4 Table S1 ΔW[High]60C (mg/ml) Self-associated species (Da) ,853 (±4,248) ,753 (±1,124) ,928 (±1,413) ,303 (±7.594) ,234 (±2,767) 4

5 Figure S1 5

6 Figure S2 6

7 Figure S3 7

8 Figure S4 8