Figure 3.

Metal-dependent aggregation. (a) Size-exclusion chromatography elution profiles of indicated constructs incubated with Cu²⁺ or Ni²⁺ present at the indicated stoichiometries. For emphasis, H13F:Ni²⁺ is shown only with gross excess of Ni²⁺. Incubation at lower stoichiometries yields similar results. In all cases, the monomer is the only detectable species if EDTA is added after ~2 h of incubation, just before size-exclusion chromatography analysis (not shown). (b) Incubation for ~2 weeks before addition of 10 mM EDTA reveals formation of Cu²⁺-free oligomeric species²⁸. (c) Kinetics of Cu²⁺-induced oligomerization were monitored as previously described¹³ using the change in fluorescence of an exogenously introduced dye, Thioflavin T¹³. Exponential profiles are observed and fit to rates of 1.1 ± 0.2 × 10⁻³ s⁻¹ and 1.1 ± 0.1 × 10⁻³ s⁻¹ for wild type (WT) and H13F, respectively. Profiles have been normalized to the tail of the exponential.