Molecular dynamics computer calculations are proposed to provide microscopically detailed relationships between the x-ray tertiary structures and the measured binding affinities in two members of the lipid binding protein superfamily. The lipid binding proteins (LBPs) are an ideal model system for studying the details of hydrophobic ligand recognition for three main reasons. First, there are a large number of LBPs with known tertiary structure. In all cases this has proven to be a beta-clam shell like structure with the ligand binding at an internal site. Second, there is excellent thermodynamic binding data available using the change in fluorescence of the acrylodan derivatized intestinal fatty acid binding protein (ADIFAB). Third, there is a rapidly increasing data set of single alanine mutants that are being characterized in terms of relative binding affinity for ligands and approached for structure determination. This allows pursuit of the proposals long-term goal: the ability to rationally predict the changes in binding affinity to a range of ligands for site-directed mutagenesis in the LBP family. This long-term goal would have important health benefits. In particular, the ability to rationally design a LBP family member with predictable binding affinity to fatty acids and/or retinoids, could lead to an important drug. These ambitious long-term goals will be approached through a series of short-term goals.
The specific aims addressing the goals are to: (1) Determine the dynamical motions and the average properties of molecular dynamics simulations of intestinal fatty acid binding protein (I-FABP) and cellular retinol binding protein II (CRBP II) structures. (2) Develop qualitative methods for the prediction of the changes in binding affinity for site-directed mutants in I-FABP and CRBP II systems. (3) Calculate lambda coupled free energy changes for subset of site-directed mutants in I-FABP and CRBP II to improve the qualitative estimates for binding affinity of the second aim. These theoretical projects will complement ongoing NIH funded experimental work in the Sacchettini, Banaszak and Kleinfeld laboratories. A dialogue between the computational and the experimental will expand the range of connections in the LBP superfamily beyond what would be possible for any group acting in isolation.
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