title: Single-molecule folding mechanism of an EF-hand neuronal calcium sensor.
abstract: EF-hand calcium sensors respond structurally to changes in intracellular Ca(2+) concentration, triggering diverse cellular responses and resulting in broad interactomes. Despite impressive advances in decoding their structure-function relationships, the folding mechanism of neuronal calcium sensors is still elusive. We used single-molecule optical tweezers to study the folding mechanism of the human neuronal calcium sensor 1 (NCS1). Two intermediate structures induced by Ca(2+) binding to the EF-hands were observed during refolding. The complete folding of the C domain is obligatory for the folding of the N domain, showing striking interdomain dependence. Molecular dynamics results reveal the atomistic details of the unfolding process and rationalize the different domain stabilities during mechanical unfolding. Through constant-force experiments and hidden Markov model analysis, the free energy landscape of the protein was reconstructed. Our results emphasize that NCS1 has evolved a remarkable complex interdomain cooperativity and a fundamentally different folding mechanism compared to structurally related proteins.
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark.
authors.forename: Pétur O
authors.initials: PO
authors.lastname: Heidarsson
authors.forename: Mariela R
authors.initials: MR
authors.lastname: Otazo
authors.forename: Luca
authors.initials: L
authors.lastname: Bellucci
authors.forename: Alessandro
authors.initials: A
authors.lastname: Mossa
authors.forename: Alberto
authors.initials: A
authors.lastname: Imparato
authors.forename: Emanuele
authors.initials: E
authors.lastname: Paci
authors.forename: Stefano
authors.initials: S
authors.lastname: Corni
authors.forename: Rosa
authors.initials: R
authors.lastname: Di Felice
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.forename: Ciro
authors.initials: C
authors.lastname: Cecconi
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D006801:Humans; D056004:Molecular Dynamics Simulation; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry*; D009479:Neuropeptides / Q000737:chemistry*; D052898:Optical Tweezers; D011485:Protein Binding; D058849:Protein Refolding; D017433:Protein Structure, Secondary
chemical_list: D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D002118:Calcium