Direct kinetic evidence for folding via a highly compact, misfolded state.
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, alpha-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 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) 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 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.
Pandya
M J
MJ
Molecular Recognition Centre and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, United Kingdom.
Williams
P B
PB
Dempsey
C E
CE
Shewry
P R
PR
Clarke
A R
AR
D055516:2S Albumins, Plant; D052179:Antigens, Plant; D006368:Helianthus; D007700:Kinetics; D008956:Models, Chemical; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D012639:Seeds / Q000737:chemistry
D055516:2S Albumins, Plant; D052179:Antigens, Plant; D010940:Plant Proteins; C067568:methionine-rich 2S protein, Helianthus