title: Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1.
abstract: Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, 2200 Copenhagen, Denmark;
authors.forename: Pétur O
authors.initials: PO
authors.lastname: Heidarsson
authors.affiliation: Institute of Nanoscience S3, Consiglio Nazionale delle Ricerche, 41125 Modena, Italy; Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, 41125 Modena, Italy;
authors.forename: Mohsin M
authors.initials: MM
authors.lastname: Naqvi
authors.affiliation: Institute of Nanoscience S3, Consiglio Nazionale delle Ricerche, 41125 Modena, Italy; Department of Physics, Center of Applied Technologies and Nuclear Development, Havana, Cuba; and.
authors.forename: Mariela R
authors.initials: MR
authors.lastname: Otazo
authors.affiliation: Department of Physics, University of Bari and Istituto Nazionale di Fisica Nucleare, Sezione di Bari, 70126 Bari, Italy.
authors.forename: Alessandro
authors.identifier: http://orcid.org/0000-0002-1589-8920
authors.initials: A
authors.lastname: Mossa
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, 2200 Copenhagen, Denmark; ciro.cecconi@gmail.com bbk@bio.ku.dk.
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.affiliation: Institute of Nanoscience S3, Consiglio Nazionale delle Ricerche, 41125 Modena, Italy; Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, 41125 Modena, Italy; ciro.cecconi@gmail.com bbk@bio.ku.dk.
authors.forename: Ciro
authors.initials: C
authors.lastname: Cecconi
mesh_terms: D002118:Calcium / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry* / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism*; D052898:Optical Tweezers; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D002118:Calcium
keywords: NCS-1; conformational dynamics; off-pathway intermediate; optical trapping; protein folding