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

• Published in: Biochemistry (Easton) 2000-02, Vol.39 (5), p.860-871
• Main Authors: Dalessio, Paula M, Ropson, Ira J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Chemical Phenomena; Chemistry, Physical; Circular Dichroism; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids - metabolism; Ileum - metabolism; Kinetics; Molecular Sequence Data; Myelin P2 Protein - chemistry; Myelin P2 Protein - metabolism; Neoplasm Proteins; Nerve Tissue Proteins; Organic Anion Transporters, Sodium-Dependent; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Rats; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Swine; Symporters
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi991937j

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-β-proteins are very similar, with 123 of the 127 amino acids of ILBP having backbone and Cβ 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.