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.
Highlights
IntroductionProteins can form partially folded, collapsed states resembling the intermediate states along the protein folding pathway, and this is important in understanding the mechanisms of protein folding [1]
The function of a protein depends on its ability to adopt a specific structure
We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies
Summary
Proteins can form partially folded, collapsed states resembling the intermediate states along the protein folding pathway, and this is important in understanding the mechanisms of protein folding [1]. Those states have been often considered as molten globule structures, that is, partially folded proteins with native-like secondary structure, but lacking the extensive, specific side-chain packing interactions of the native structure [2]. The protein in the pre-molten globule state is half-way between the molten globule and the unfolded state, it is less compact than the PLOS ONE | DOI:10.1371/journal.pone.0158430. The protein in the pre-molten globule state is half-way between the molten globule and the unfolded state, it is less compact than the PLOS ONE | DOI:10.1371/journal.pone.0158430 July 5, 2016
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