Characterization of the Molten Globule of Human Serum Retinol-Binding Protein Using NMR Spectroscopy^†,‡

The molten globule state is a partially folded conformer of proteins that has been the focus of intense study for more than two decades. This non-native fluctuating conformation has been linked to protein-folding intermediates, to biological function, and more recently to precursors in amyloid fibril formation. The molten globule state of human serum retinol-binding protein (RBP) has been postulated previously to be involved in the mechanism of ligand release (Ptitsyn, O. B., et al. (1993) FEBS Lett. 317, 181−184). Conserved residues within RBP have been identified and proposed to be key to folding and stability, although a link to a molten globule state has not previously been shown (Greene, L. H., et al. (2003) FEBS Lett. 553, 39−44). In this work, a detailed characterization of the acid-induced molten globule of RBP is presented. Using stopped-flow fluorescence spectroscopy in the presence of 8-anilino-1-naphthalene sulfonic acid (ANS), we show that RBP populates a state with molten-globule-like characteristics early in refolding. To gain insight into the structural features of the molten globule of RBP, we have monitored the denaturant-induced unfolding of this ensemble using NMR spectroscopy. The transition at the level of individual residues is significantly more cooperative than that found previously for the archetypal molten globule, α-lactalbumin (α-LA); this difference may be due to a predominantly β-sheet structure present in RBP in contrast to the α-helical nature of the α-LA molten globule.