PMID:22060017
| Citation |
Belevich, G, Knuuti, J, Verkhovsky, MI, Wikström, M and Verkhovskaya, M (2011) Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis. Mol. Microbiol. 82:1086-95 |
|---|---|
| Abstract |
The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed. |
| Links |
PubMed PMC3274701 Online version:10.1111/j.1365-2958.2011.07883.x |
| Keywords |
Amino Acid Substitution; Electron Transport Complex I/genetics; Electron Transport Complex I/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Models, Biological; Models, Molecular; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Mutant Proteins/genetics; Mutant Proteins/metabolism; NADH Dehydrogenase/genetics; NADH Dehydrogenase/metabolism; Protein Subunits/genetics; Protein Subunits/metabolism; Sequence Deletion |
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