title: Unfolding studies of tissue transglutaminase.
abstract: Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Interdisciplinary Centre for Study of Inflammation, University of Ferrara, Via L. Borsari 46, 44100, Ferrara, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Cervellati
authors.forename: Lorella
authors.initials: L
authors.lastname: Franzoni
authors.forename: Monica
authors.initials: M
authors.lastname: Squerzanti
authors.forename: Carlo M
authors.initials: CM
authors.lastname: Bergamini
authors.forename: Francesco
authors.initials: F
authors.lastname: Spinozzi
authors.forename: Paolo
authors.initials: P
authors.lastname: Mariani
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Lanzara
authors.forename: Alberto
authors.initials: A
authors.lastname: Spisni
mesh_terms: D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013997:Time Factors; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D019791:Guanidine; D002118:Calcium