FOLDING MECHANISM OF 3 STRUCTURALLY SIMILAR BETA-SHEET PROTEINS

The folding mechanism of cellular retinoic acid binding protein I (CRA BP I), cellular retinol binding protein II (CRBP II), and intestinal f atty acid binding protein (IFABP) were investigated to determine if pr oteins with similar native structures have similar folding mechanisms. These mostly beta-sheet proteins have very similar structures, despit e having as little as 33% sequence similarity, The reversible urea den aturation of these proteins was characterized at equilibrium by circul ar dichroism and fluorescence. The data were best fit by a two-state m odel for each of these proteins, suggesting that no significant popula tion of folding intermediates were present at equilibrium, The native states were of similar stability with free energies (linearly extrapol ated to 0 M urea, Delta G(H2O)) of 6.5, 8.3, and 5.5 kcal/mole for CRA BP I, CRBP II, and IFABP, respectively. The kinetics of the folding an d unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the fold ing and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude . Further, the spectroscopic properties of the intermediate states wer e different for each protein, suggesting that different amounts of sec ondary and/or tertiary structure were associated with each intermediat e state for each protein. These data show that the folding path for pr oteins in the same structural family can be quite different, and provi de evidence for different folding landscapes for these sequences. (C) 1998 Wiley-Liss, Inc.