title: Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C.
abstract: The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.
authors.affiliation: Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306-4300, USA.|Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas F° 373, Cidade Universitária, Rio de Janeiro, 21941-902, RJ, Brazil.
authors.forename: Tiago
authors.initials: T
authors.lastname: Veltri
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Guilherme A P
authors.initials: GAP
authors.lastname: de Oliveira
authors.affiliation: Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306-4300, USA.
authors.forename: Ewa A
authors.initials: EA
authors.lastname: Bienkiewicz
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas F° 373, Cidade Universitária, Rio de Janeiro, 21941-902, RJ, Brazil.
authors.forename: Fernando L
authors.initials: FL
authors.lastname: Palhano
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Mayra de A
authors.initials: MA
authors.lastname: Marques
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.|Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Brazil.
authors.forename: Adolfo H
authors.initials: AH
authors.lastname: Moraes
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Jerson L
authors.identifier: http://orcid.org/0000-0001-9523-9441
authors.initials: JL
authors.lastname: Silva
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas F° 373, Cidade Universitária, Rio de Janeiro, 21941-902, RJ, Brazil.
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
authors.affiliation: Department of Biomedical Sciences, Florida State University College of Medicine, 1115 West Call Street, Tallahassee, FL, 32306-4300, USA. jose.pinto@med.fsu.edu.
authors.forename: Jose R
authors.identifier: http://orcid.org/0000-0001-9092-4976
authors.initials: JR
authors.lastname: Pinto
mesh_terms: D000483:Alleles; D000577:Amides / Q000737:chemistry*; D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000577:Amides; D019209:Troponin C; D006859:Hydrogen; D002118:Calcium