title: Single-molecule folding mechanisms of the apo- and Mg(2+)-bound states of human neuronal calcium sensor-1.
abstract: Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca(2+) concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca(2+)-bound state, the presence of Mg(2+) allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.
authors.affiliation: Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, Modena, Italy; CNR Institute of Nanoscience S3, Modena, Italy.
authors.forename: Mohsin M
authors.initials: MM
authors.lastname: Naqvi
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Pétur O
authors.initials: PO
authors.lastname: Heidarsson
authors.affiliation: CNR Institute of Nanoscience S3, Modena, Italy; Department of Physics, Center of Applied Technologies and Nuclear Development (CEADEN), Miramar, La Habana, Cuba.
authors.forename: Mariela R
authors.initials: MR
authors.lastname: Otazo
authors.affiliation: Department of Physics, University of Bari and INFN, Sezione di Bari, Bari, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Mossa
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen N, Denmark. Electronic address: bbk@bio.ku.dk.
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.affiliation: Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, Modena, Italy; CNR Institute of Nanoscience S3, Modena, Italy. Electronic address: ciro.cecconi@gmail.com.
authors.forename: Ciro
authors.initials: C
authors.lastname: Cecconi
mesh_terms: D002413:Cations, Divalent / Q000737:chemistry; D003198:Computer Simulation; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008390:Markov Chains; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry*; D009479:Neuropeptides / Q000737:chemistry*; D052898:Optical Tweezers; D017510:Protein Folding; D013057:Spectrum Analysis; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D008274:Magnesium