FIGURE 8.

Mutagenesis of basic residues within central LRR barrel. The 12 Arg/Lys residues mutated to Glu to assess their involvement in LC1-microtubule interactions are indicated in the top left panel. These residues form two clusters: a group near the N-terminal end of the central barrel and a helical stripe that extends along the barrel and crosses the protein short axis. The conservation of these residues between Chlamydomonas and humans is mapped onto the structure in the top right panel. The color code is as follows: identical, deep salmon; conservative substitution, olive; not conserved, green. Part of the N-terminal basic patch and much of the helical stripe is highly conserved. The lower panel shows the gel filtration profiles for K8E, K18E, and R79E forms of LC1 that exhibit distinct propensities for oligomerization: the peak eluting at ∼16 ml represents monomeric LC1, whereas oligomers elute earlier. The insets show the final purified mutant proteins separated in 12.5% SDS-polyacrylamide gels (Coomassie Blue stain).