title: Conformational stability of human skeletal tropomyosins modified by site-directed mutagenesis.
abstract: We have used human beta-tropomyosin produced in Escherichia coli and deletion mutants obtained by site-directed mutagenesis to analyse the conformational stability of this molecule under various experimental conditions. Protein engineering has allowed us to answer some questions raised by stability analysis of the wild-type tropomyosin. The complex pattern of denaturation is due neither to heterogeneity of the preparation nor to head-to-tail interactions. The N- and C-termini are not of importance for the thermal stability of the molecule. On the contrary, deletion of the 31 C-terminus amino acids leads to a dramatic decrease of the stability observed in guanidinium chloride. This lowering is interpreted as the participation of one more guanidinium chloride ions to the denaturation equilibrium. Analysis of the stability in presence of organic solvents reveals that acetonitrile and methanol induce opposite effects. Investigation of these effects by three methods (CD, fluorescence and electrophoresis that measure respectively the content in alpha-helix, the contact between the two strands and the strands exchange) leads to the conclusion that strand separation can precede the denaturation of the alpha-helix.
authors.affiliation: INSERM U-249, CNRS, Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Ferraz
authors.forename: F
authors.initials: F
authors.lastname: Heitz
authors.forename: J
authors.initials: J
authors.lastname: Sri Widada
authors.forename: E
authors.initials: E
authors.lastname: Caron
authors.forename: A
authors.initials: A
authors.lastname: Cave
authors.forename: J P
authors.initials: JP
authors.lastname: Liautard
mesh_terms: D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics
chemical_list: D006146:Guanidines; D012997:Solvents; D014335:Tropomyosin; D019791:Guanidine