title: Structural stabilities of different regions of the titin I27 domain contribute differently to unfolding upon mitochondrial protein import.
abstract: Protein import into mitochondria requires unfolding of the folded mature domain of precursor proteins. Here we compared the effects of amino-acid replacement between the core region and the N-terminal region of the titin I27 domain (the 27th Ig domain of human titin) on its import into isolated mitochondria when attached to a short presequence (pb(2)(35)). We found that several mutations in the core region around Trp34 of the I27 domain enhanced the import rates of the fusion proteins, while the N-terminal K6P mutation, which increases mechanical stability around the N-terminal region, decreases the import rate. When the K6P mutation is combined with core-destabilizing mutations, the import rates of the fusion proteins still decrease, unless a long segment is deleted. These results suggest that mutations in the core region could destabilize the transition state for unfolding from the intermediate with the detached N-terminal segment during import, leading to enhanced unfolding rates, although stabilization of the N-terminal region masks these effects. In other words, the rate-limiting step of the global unfolding upon import into mitochondria switches, depending on the balance between the stability of the N-terminal structure and the stability of the core region of the I27 domain.
authors.affiliation: Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Takaomi
authors.initials: T
authors.lastname: Oguro
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yagawa
authors.forename: Takaki
authors.initials: T
authors.lastname: Momose
authors.forename: Takehiro
authors.initials: T
authors.lastname: Sato
authors.forename: Koji
authors.initials: K
authors.lastname: Yamano
authors.forename: Toshiya
authors.initials: T
authors.lastname: Endo
mesh_terms: D002942:Circular Dichroism; D064211:Connectin; D006801:Humans; D018625:Microscopy, Atomic Force; D008928:Mitochondria / Q000378:metabolism*; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D013050:Spectrometry, Fluorescence
chemical_list: D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases