title: The Y9P Variant of the Titin I27 Module: Structural Determinants of Its Revisited Nanomechanics.
abstract: The titin I27 module from human cardiac titin has become a standard in protein nanomechanics. A proline-scanning study of its mechanical clamp found three mechanically hypomorphic mutants and a paradoxically hypermorphic mutant (I27Y9P). Both types of mutants have been commonly used as substrates of several protein unfoldase machineries in studies relating protein mechanostability to translocation or degradation rates. Using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and steered molecular dynamics simulations, we show that, unexpectedly, the mechanostability of the Y9P variant is comparable to the wild type. Furthermore, the NMR analysis of homomeric polyproteins of this variant suggests that these constructs may induce slight structural perturbations in the monomer, which may explain some minor differences in this variant's properties; namely the abolishment of the mechanical unfolding intermediate and a reduced thermal stability. Our results clarify a previously reported paradoxical result in protein nanomechanics and contribute to refining our toolbox for understanding the unfolding mechanism used by translocases and degradation machines.
authors.affiliation: Instituto Cajal, IC-CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain; Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 28049 Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Oroz
authors.affiliation: Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
authors.affiliation: Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
authors.affiliation: Instituto Cajal, IC-CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain; Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 28049 Madrid, Spain.
authors.forename: Albert
authors.initials: A
authors.lastname: Galera-Prat
authors.affiliation: Centro Nacional de Biotecnología, CSIC, Darwin, 28049 Madrid, Spain.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schönfelder
authors.affiliation: Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Cañada
authors.affiliation: Instituto Cajal, IC-CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain; Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 28049 Madrid, Spain. Electronic address: mcarrion@cajal.csic.es.
authors.forename: Mariano
authors.initials: M
authors.lastname: Carrión-Vázquez
mesh_terms: D064211:Connectin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D020815:Polyproteins / Q000378:metabolism*; D011392:Proline / Q000235:genetics*; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D000072760:Single Molecule Imaging; D014443:Tyrosine / Q000235:genetics*
chemical_list: D064211:Connectin; D020815:Polyproteins; C577285:TTN protein, human; D014443:Tyrosine; D011392:Proline