All three tryptophan residues in alpha-subunit of mitochondrial processing peptidase (MPP) were subsequently substituted. While substitutions of Trp223 led to misfolded non-functional protein, mutations of Trp147 and/or Trp481 did not affect the enzyme processing activity. Thus, fluorescence properties of the mutants with fewer tryptophans were used for observation of both alpha-MPP domain translocation and visualization of conformational changes in the interdomain linker evoked by substrate. We found that in the presence of substrate the C-terminal penultimate Trp481 was approaching Trp223, which is localized at the border of N-terminal domain and interdomain linker. Also, excision of the alpha-MPP C-terminal 30 amino acid residues (DeltaC30) led to a complete loss of protein function. Even shorter deletions of the alpha-MPP C-terminus destabilized the protein slightly (DeltaC2) or dramatically (DeltaC17). It suggests that the extreme C-terminus of alpha-MPP provides mechanical support to the C-terminal domain during its extensive conformational change accompanying the substrate recognition process.

Institute of Microbiology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague 4 Czech Republic

References provided by Crossref.org

An Advanced System of the Mitochondrial Processing Peptidase and Core Protein Family in Trypanosoma brucei and Multiple Origins of the Core I Subunit in Eukaryotes

A computational study of the glycine-rich loop of mitochondrial processing peptidase

Reductive evolution of the mitochondrial processing peptidases of the unicellular parasites trichomonas vaginalis and giardia intestinalis