J Biol Chem, Vol. 274, Issue 38, 26828-26837, September 17, 1999

Direct Kinetic Evidence for Folding via a Highly Compact, Misfolded State

Maya J. Pandya^§, Phil B. Williams, Christopher E. Dempsey, Peter R. Shewry^§, and Anthony R. Clarke

From the  Molecular Recognition Centre and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom and ^§ Institute of Arable Crops Research, Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS18 9AF, United Kingdom

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, -helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m_U (4.7 M¹ at 10 °C) and C_p (0.95 kcal mol¹ K¹) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m_M = 0.95 M¹ at 10 °C) is more compact than the transition state for folding (m_T = 2.5 M¹ at 10 °C), provides direct kinetic evidence for the transient misfolding of a protein.