title: Ligand-dependent equilibrium fluctuations of single calmodulin molecules.
abstract: Single-molecule force spectroscopy allows superb mechanical control of protein conformation. We used a custom-built low-drift atomic force microscope to observe mechanically induced conformational equilibrium fluctuations of single molecules of the eukaryotic calcium-dependent signal transducer calmodulin (CaM). From this data, the ligand dependence of the full energy landscape can be reconstructed. We find that calcium ions affect the folding kinetics of the individual CaM domains, whereas target peptides stabilize the already folded structure. Single-molecule data of full length CaM reveal that a wasp venom peptide binds noncooperatively to CaM with 2:1 stoichiometry, whereas a target enzyme peptide binds cooperatively with 1:1 stoichiometry. If mechanical load is applied directly to the target peptide, real-time binding/unbinding transitions can be observed.
authors.affiliation: Physik Department E22, Technische Universität München, James-Franck-Strasse, 85748 München, Germany.
authors.forename: Jan Philipp
authors.initials: JP
authors.lastname: Junker
authors.forename: Fabian
authors.initials: F
authors.lastname: Ziegler
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D018625:Microscopy, Atomic Force; D009010:Monte Carlo Method; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D014862:Wasp Venoms; C037500:mastoparan; D009219:Myosin-Light-Chain Kinase; D002118:Calcium