Complex formation and kinetics of filament assembly exhibited by the simple epithelial keratins K8 and K18

We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8 M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2 s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length lp of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl2 increases lp to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.