The folding mechanisms of two proteins in the family of intracellular lipid binding proteins, ileal lipid binding protein (ILBP) and intestinal fatty acid binding protein (IFABP), were examined. The structures of these all-beta-proteins are very similar, with 123 of the 127 amino acids of ILBP having backbone and C(beta) conformations nearly identical to those of 123 of the 131 residues of IFABP. Despite this structural similarity, the sequences of these proteins have diverged, with 23% sequence identity and an additional 16% sequence similarity. The folding process was completely reversible, and no significant concentrations of intermediates were observed by circular dichroism or fluorescence at equilibrium for either protein. ILBP was less stable than IFABP with a midpoint of 2. 9 M urea compared to 4.0 M urea for IFABP. Stopped-flow kinetic studies showed that both the folding and unfolding of these proteins were not monophasic, suggesting that either multiple paths or intermediate states were present during these processes. Proline isomerization is unlikely to be the cause of the multiphasic kinetics. ILBP had an intermediate state with molten globule-like spectral properties, whereas IFABP had an intermediate state with little if any secondary structure during folding and unfolding. Double-jump experiments showed that these intermediates appear to be on the folding path for each protein. The folding mechanisms of these proteins were markedly different, suggesting that the different sequences of these two proteins dictate different paths through the folding landscape to the same final structure.

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly beta-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, deltaGH2O) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding 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 were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences.

Although large changes in fluorescence intensity are observed during the folding and unfolding of many proteins, it has been difficult to associate these changes with specific structures or with the environmental changes which a particular tryptophan may undergo during these processes. The fluorescence spectral changes that occur during the folding and unfolding of rat intestinal fatty acid binding protein (IFABP) are described here. The intermediate observed during unfolding had spectral characteristics similar to those of unfolded protein, but with somewhat higher intensity. Stopped-flow circular dichroism measurements during unfolding showed that little if any secondary structure was associated with this intermediate. During refolding, the initial fluorescence spectrum was not that of native or unfolded IFABP, suggesting that some structure with intermediate fluorescent properties had formed during the deadtime of mixing. The shape and intensity of this initial spectrum were dependent on the final urea concentration, becoming more native-like at lower final concentrations of denaturant. A simple model for refolding suggests that a portion of the protein molecules obtain native structure and fluorescent characteristics during the deadtime of mixing, and that the remaining protein molecules have spectral characteristics similar to those of the intermediate observed during unfolding. Lower final concentrations of denaturant cause a larger proportion of molecules to follow the rapid refolding pathway. Knowledge of the fluorescence spectral characteristics of the intermediates formed during the folding and unfolding of any protein will improve our understanding of the nature of these structures.