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, alpha-lactalbumin (alpha-LA); this difference may be due to a predominantly beta-sheet structure present in RBP in contrast to the alpha-helical nature of the alpha-LA molten globule.
Greene
Lesley H
LH
Department of Biochemistry and Chemistry Research Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Wijesinha-Bettoni
Ramani
R
Redfield
Christina
C
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D017124:Conserved Sequence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012177:Retinol-Binding Proteins / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D001798:Blood Proteins; D012177:Retinol-Binding Proteins; C027132:1-anilino-8-naphthalenesulfonate