Supplementary material for Botuyan et al. (1999) Proc. Natl. Acad. Sci. USA 96(16), 9033-9038.

Sedimentation velocity data at 40,000 rpm for Elc1 (yeast homologue of elongin C) and mixtures of Elc1 and VHL (von Hippel-Lindau) peptide (4 mg/ml) were subjected to the analysis method of Stafford [Stafford, W. (1992) Anal. Biochem. 203, 295-301] to calculate the apparent sedimentation coefficient distributions {g(s*)}.

The apparent sedimentation coefficient distributions for Elc1 exhibited a relatively narrow distribution (Fig. 6A). Contributions decreased steadily on either side of 1.2 S. The results suggest the presence of predominantly one species. Estimation of the diffusion constant from the width of the distribution at half the maximum height enabled measurement of the apparent molecular mass of the sedimenting species, which was 44 kDa. Given that the sequence molecular mass of Elc1 is 11.8 kDa, the results suggest that Elc1 sediments as a tetramer. The apparent sedimentation coefficient distributions for mixtures of Elc1 and VHL peptide also exhibited a relatively narrow distribution (Fig. 6B), suggesting the presence of predominantly one species. However, estimation of the apparent molecular mass of the sedimenting species revealed a value of 28.4 kDa. This apparent molecular mass is close to the expected molecular mass of dimeric Elc1 bound to two VHL peptides.

Fig. 6. Apparent sedimentation coefficient distribution {g(s*)} as a function of sedimentation coefficient. Sedimentation velocity data at 40,000 rpm over the time range of 500-1000 s (total of 18 scans) were fit by using the time derivative analysis module of the Beckman XLI data analysis software. (A) Elc1. (B) Mixture of Elc1 and VHL peptide.