title: Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin.
abstract: Stopped-flow kinetic and fluorescence spectroscopic analyses, including solvent and temperature perturbations, of five isofunctional structural mutants of calmodulin indicate that calcium binding to calmodulin follows the order site III, site IV, site I, site II, with dissociation occurring in the reverse order. Each of the isofunctional structural mutants contains a single tryptophan residue, introduced by site-specific mutagenesis, as an internal spectroscopic reporter group that was used as a probe of local conformational change. Calcium binding was studied by using flow dialysis or by using fluorescence spectroscopy and monitoring the change in the single tryptophan residue in each calcium-binding site. Calcium removal was examined by using EDTA and monitoring tryptophan fluorescence or by using Quin 2 and monitoring the change in the chromophoric chelator. Computational analysis of the data suggests a rate-limiting step for dissociation between calcium removal from sites I/II and sites III/IV. Unexpected results with the site IV isofunctional mutant (Q135W-CaM) indicated cross-talk between the amino and carboxyl terminal halves of CaM during the calcium-binding mechanism. Studies with ethylene glycol provided empirical data that suggest the functional importance of the electrostatic potential of CaM, or the molarity of water, in the calcium-binding process. Altogether, the data allowed a kinetic extension of the sequential, cooperative model for calcium binding to calmodulin and provided values for additional parameters in the model of calcium binding to CaM, a prototypical member of the family of proteins required for calcium signal transduction in eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratoire de Biophysique, Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France.
authors.forename: M C
authors.initials: MC
authors.lastname: Kilhoffer
authors.forename: M
authors.initials: M
authors.lastname: Kubina
authors.forename: F
authors.initials: F
authors.lastname: Travers
authors.forename: J
authors.initials: J
authors.lastname: Haiech
mesh_terms: D000595:Amino Acid Sequence; D000634:Aminoquinolines; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D019855:Ethylene Glycol; D005026:Ethylene Glycols / Q000494:pharmacology; D005454:Fluorescence Polarization; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D000634:Aminoquinolines; D002147:Calmodulin; D005026:Ethylene Glycols; D012997:Solvents; D014364:Tryptophan; D019855:Ethylene Glycol; C035739:Quin2; D002118:Calcium