To validate the model for AmiC and its interactions with CNP and AmiC (main paper Figure 3), we investigated the stoichiometry of AmiC using both native gel electrophoresis and analytical gel filtration. Native gel electrophoresis (Supplemental Figure S3A) showed that AmiC behaves as a protein larger than the expected 45 kDa. Fitting of the molecular weight standards suggests a molecular weight for AmiC of 120 ± 40 kDa. As AmiC has more negative charges than positive charges, it would be expected to run slightly anomalously quickly rather than slowly. Analytical size exclusion chromatography showed that AmiC behaves as expected for a spherical protein with a molecular weight of 136 ± 3 kDa (Supplemental Figure S3B). These data strongly suggest that AmiC natively adopts an oligomeric state, as the protein alone is roughly spherical and has no unusual sequences. These methods both largely reflect the Stoke’s radius of the protein rather than molecular weight: consequently, it is likely that proteins will behave as a larger spherical protein than their true molecular weight. The results obtained would be consistent with either a non-spherical dimer of AmiC, or an almost perfectly spherical trimer of AmiC. Given the predicted dimeric state from crystal structures, the lack of a realistic trimeric structure in these structures, and the highly non-spherical native of the putative dimer, the most likely interpretation of these results is that AmiC natively forms a dimer.
Fig S3A AmiC forms a dimer on native gel electrophoresis. AmiC was run on a native gel with standard markers. The mobilities of the standard proteins were fitted to a standard logarithmic equation using GraphPad Prism, showing an excellent fit to the data (R2 = 0.97). Using this fitting, the molecular weight of AmiC was calculated. The data shown are representative of three independent experiments, performed on separate days.
Fig. S3B Analytical size exclusion of AmiC. AmiC and standard proteins were analysed on an analytical gel filtration column. The peaks of elution obtained were fitted to a standard equation using GraphPad Prism, showing an excellent fit to the data. The molecular weight of AmiC was then calculated using this equation. The results shown are the means of at least four independent experiments conducted over at least two days.
Methods:
Native gel electrophoresis
The native PAGE 12-4% Tris Glycine (Amersham ECL, GE Healthcare) gels were run at 160 V for 1.6h at room temperature in 25 mM Tris, 192 mM glycine, pH 8.3. The NativeMark™ Unstained Protein Standard (Life Technologies) was used as a molecular maker and the relative molecular distances calculated by diving the migration distance of the protein by the migration of the dye front. A log of the molecular weight was plotted versus the Rf values and the molecular weight of AmiC determined by interpolating unknowns from the standard curve using linear regression (GraphPad Prism version 5).
Analytical size-exclusion chromatography
Size-exclusion chromatography was performed using a Superdex 200 10/300 GL column (GE Healthcare; 24 mL bed volume) attached to an ÄKTApurifier system (GE Healthcare). 100 mL samples were loaded into a 200 mL loop, injected onto the column, and eluted isocratically at a flow rate of 0.5 ml min-1 with 10 mM Hepes-NaOH pH 7.5, 150 mM NaCl. Absorbance of the eluent was monitored at 280 nm; the elution volume was determined as the point of highest absorbance (all samples showed approximately symmetrical absorbance peaks). The column was calibrated by applying in the same manner 100 ml of Apoferritin (443 kDa), β-Amylase (200 kDa), Alcohol Dehydrogenase (150 kDa, Albumin (66 kDa), Ovalbumin (44 kDa), Carbonic Anhydrase (29 kDa) and BpML1 (11 kDa)1. For each sample, at least four independent experiments were performed on at least two days; standards were paired with samples eluting at very different volumes run together. AmiC molecular mass was calculated using the following formula:
Log10RMM = (m * elution vol.) + C
Values for the constants m and C were determined from the data by linear regression using GraphPad Prism version 5.
1 Norville, I. H. et al. (2011) The structure of a Burkholderia pseudomallei immunophilin-inhibitor complex reveals new approaches to antimicrobial development. Biochem. J. 437, 413-422.