Current efforts in this laboratory are focused on the folding, stability and function of a large family of all ß-sheet proteins, the intracellular lipid binding proteins. These proteins have very similar structures but diverse amino-acid sequences and ligand specificities and are crucial to hydrophobic ligand transport and utilization within the cell. We have shown that there are at least two intermediates present on the folding pathway for one of these proteins, intestinal fatty-acid binding protein (IFABP). These intermediates involve only one of the two tryptophans in the sequence of IFABP, and may act as folding initiation sites. Studies on other members of the family have shown surprising heterogeneity in the mechanism of folding for these structurally similar proteins. The overall rates of folding and unfolding vary by as much as 4 orders of magnitude for different members of the family. Further, the spectroscopic properties of the intermediates are generally not similar among these proteins, implying that the intermediate structures responsible for these spectroscopic properties are also different. We are using nuclear magnetic resonance, small angle x-ray scattering, fluorescence and circular dichroism to better describe the size, shape, and spectroscopic properties of these intermediates, both kinetically and at equilibrium. Little is known about the folding path or the mechanism of stabilization of any ß-sheet protein, emphasizing the importance of these studies to a general understanding of the protein folding problem.
A related project is to determine the physical forces that stabilize this structural motif. This project uses site-directed mutagenesis and molecular dynamics calculations to determine the importance of hydrophobic packing, hydrogen bonding patterns, and buried charge neutralization to the overall stability of these ß-sheet proteins. The overall structure of these proteins is unusual, since there is a large internal solvent filled cavity in the apo-structures of all of these proteins, which is only partially filled by ligand after binding. The overall structure of the protein backbone changes very little when ligand binds, providing no obvious means of entry of the ligand to this buried pocket.
Several proteins in this family are phosphorylated on a tyrosine by the insulin receptor, which suggests an additional regulatory function for these proteins. Phosphorylation may cause changes in overall protein stability, ligand affinity, and/or ligand flux within the cell. These proteins could serve as models for tyrosine phosphorylation and its effects on protein structure, which has important implications for gene regulation since so many regulatory proteins are tyrosine phosphorylated. There is evidence that one of these proteins may act as inhibitory growth factors for breast cancer cells in vitro, again suggesting an additional role for these proteins in the regulation of cell proliferation.