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Comparison of the protein-unfolding pathways between mitochondrial protein import and atomic-force microscopy measurements.

ABSTRACT: Many newly synthesized proteins have to become unfolded during translocation across biological membranes. We have analyzed the effects of various stabilization/destabilization mutations in the Ig-like module of the muscle protein titin upon its import from the N terminus or C terminus into mitochondria. The effects of mutations on the import of the titin module from the C terminus correlate well with those on forced mechanical unfolding in atomic-force microscopy (AFM) measurements. On the other hand, as long as turnover of the mitochondrial Hsp70 system is not rate-limiting for the import, import of the titin module from the N terminus is sensitive to mutations in the N-terminal region but not the ones in the C-terminal region that affect resistance to global unfolding in AFM experiments. We propose that the mitochondrial-import system can catalyze precursor-unfolding by reducing the stability of unfolding intermediates.

SUBMITTER: Sato T

PROVIDER: S-EPMC1312372 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Comparison of the protein-unfolding pathways between mitochondrial protein import and atomic-force microscopy measurements.

Sato Takehiro T Esaki Masatoshi M Fernandez Julio M JM Endo Toshiya T

Proceedings of the National Academy of Sciences of the United States of America 20051202 50

Many newly synthesized proteins have to become unfolded during translocation across biological membranes. We have analyzed the effects of various stabilization/destabilization mutations in the Ig-like module of the muscle protein titin upon its import from the N terminus or C terminus into mitochondria. The effects of mutations on the import of the titin module from the C terminus correlate well with those on forced mechanical unfolding in atomic-force microscopy (AFM) measurements. On the other ...[more]

PMID: 16326810

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Project description:Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.

Dataset Information

Comparison of the protein-unfolding pathways between mitochondrial protein import and atomic-force microscopy measurements.

Publications

Comparison of the protein-unfolding pathways between mitochondrial protein import and atomic-force microscopy measurements.

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