title: Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase.
abstract: Tissue transglutaminase undergoes thermal inactivation with first-order kinetics at moderate temperatures, in a process which is affected in opposite way by the regulatory ligands calcium and GTP, which stabilize different conformations. We have explored the processes of inactivation and of unfolding of transglutaminase and the effects of ligands thereon, combining approaches of differential scanning calorimetry (DSC) and of thermal analysis coupled to fluorescence spectroscopy and small angle scattering. At low temperature (38-45°C), calcium promotes and GTP protects from inactivation, which occurs without detectable disruption of the protein structure but only local perturbations at the active site. Only at higher temperatures (52-56°C), the protein structure undergoes major rearrangements with alterations in the interactions between the N- and C-terminal domain pairs. Experiments by DSC and fluorescence spectroscopy clearly indicate reinforced and weakened interactions of the domains in the presence of GTP and of calcium, and different patterns of unfolding. Small angle scattering experiments confirm different pathways of unfolding, with attainment of limiting values of gyration radius of 52, 60 and 90 Å in the absence of ligands and in the presence of GTP and calcium. Data by X-rays scattering indicate that ligands influence retention of a relatively compact structure in the protein even after denaturation at 70°C. These results suggest that the complex regulation of the enzyme by ligands involves both short- and long-range effects which might be relevant for understanding the turnover of the protein in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Ferrara, Via Borsari 46, 44100, Ferrara, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Cervellati
authors.forename: Katy
authors.initials: K
authors.lastname: Montin
authors.forename: Monica
authors.initials: M
authors.lastname: Squerzanti
authors.forename: Carlo
authors.initials: C
authors.lastname: Mischiati
authors.forename: Carlo
authors.initials: C
authors.lastname: Ferrari
authors.forename: Francesco
authors.initials: F
authors.lastname: Spinozzi
authors.forename: Paolo
authors.initials: P
authors.lastname: Mariani
authors.forename: Heinz
authors.initials: H
authors.lastname: Amenitsch
authors.forename: Carlo M
authors.initials: CM
authors.lastname: Bergamini
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Lanzara
mesh_terms: D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D008024:Ligands; D006160:Guanosine Triphosphate; D011503:Transglutaminases; D002118:Calcium