title: Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B.
abstract: Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040, Madrid, Spain.
authors.forename: Ángeles
authors.initials: Á
authors.lastname: Bañares-Hidalgo
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040, Madrid, Spain.
authors.forename: Jesús
authors.identifier: http://orcid.org/0000-0003-3587-7147
authors.initials: J
authors.lastname: Pérez-Gil
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040, Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Estrada
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D057446:Hydrodynamics; D007700:Kinetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011510:Proteolipids / Q000737:chemistry*; D037701:Pulmonary Surfactant-Associated Protein B / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D011498:Protein Precursors; D011510:Proteolipids; D037701:Pulmonary Surfactant-Associated Protein B; C105511:surfactant protein B propeptide; D012965:Sodium Chloride; D014508:Urea; D019791:Guanidine