β-Sheet Proteins with Nearly Identical Structures Have Different Folding Intermediates

Abstract

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 search for local native-like nucleation centers in the unfolded state of β-sheet proteins

“…3 (second row), seven local native-like nucleation centers, shown in green, may be identified for ILBP roughly corresponding to the ␣-␣ turn, and the ␤-turns BC, CD, EF, FG, GH, and IJ. The nascent ␤-turns may rapidly develop into six full-fledged ␤-hairpins, forming an intermediate that may show CD spectra very similar to those of the native structure (eight ␤-hairpins total) in agreement with experimental observations (21). However, the local nucleation center in the region of the DE ␤-turn present in IFABP is notably absent in ILBP, ruling out early formation of a hydrophobic interaction that may be important for stabilization of the native structure (see discussion above and in ref.…”

“…The computational procedure found all lowenergy conformers of peptide backbones for all of the 126 hexapeptide fragments that together comprise the entire IFABP sequence. The numbers of low-energy conformers per fragment varied from 4 (fragment 113-118) to 407 (fragment [19][20][21][22][23][24] with the average value of Ϸ87. For each hexapeptide, the number of low-energy conformations geometrically similar to the 3D structure of the corresponding hexapeptide fragment in the known x-ray structure of IFABP (PDB entry 1IFC) has been determined.…”

“…[18][19][20][21][22][23]. The native 3D structures of these four proteins are very similar [the PDB entries are 1IFC (IFABP), 1EIO (ILBP), 1CBI (CRABP I), and 1OPA (CRPB II), respectively], although they possess as little as Ϸ30% sequence similarity (21,24).…”

“…Earlier data suggested that the initial step in IFABP folding is hydrophobic collapse bringing together residues belonging to the C, D, E, F, and G ␤-strands (18), a view developed further in a recent publication (22). A somewhat different model has been proposed for ILBP (21). The authors suggest that ILBP is less stable than IFABP and proceeds through formation of a molten globule-like structure rather than through a relatively limited collapse of hydrophobic clusters, as IFABP does.…”

Gene Structure and M20T Polymorphism of theSchistosoma mansoni Sm14 Fatty Acid-binding Protein

“…5A). It is interesting to note that despite the fact that distinct fatty acid-binding proteins have a similar ␤-barrel structure, they may have different folding and unfolding intermediates (20). From the fitting of the CD traces recorded at an increasing temperature (Fig.…”