David S. Riddle

Experiment and theory highlight role of native state topology in SH3 folding

We use a combination of experiments, computer simulations and simple model calculations to characterize, first, the folding transition state ensemble of the src SH3 domain, and second, the features of the protein that determine its folding mechanism. Kinetic analysis of mutations at 52 of the 57 residues in the src SH3 domain revealed that the transition state ensemble is even more polarized than suspected earlier: no single alanine substitution in the N-terminal 15 residues or the C-terminal 9 residues has more than a two-fold effect on the folding rate, while such substitutions at 15 sites in the central three-stranded β-sheet cause significant decreases in the folding rate. Molecular dynamics (MD) unfolding simulations and ab initio folding simulations on the src SH3 domain exhibit a hierarchy of folding similar to that observed in the experiments. The similarity in folding mechanism of different SH3 domains and the similar hierarchy of structure formation observed in the experiments and the simulations can be largely accounted for by a simple native state topology-based model of protein folding energy landscapes.

Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain

Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two β-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.

Simplified proteins: minimalist solutions to the 'protein folding problem'.

Recent research has suggested that stable, native proteins may be encoded by simple sequences of fewer than the full set of 20 proteogenic amino acids. Studies of the ability of simple amino acid sequences to encode stable, topologically complex, native conformations and to fold to these conformations in a biologically relevant time frame have provided insights into the sequence determinants of protein structure and folding kinetics. They may also have important implications for protein design and for theories of the origins of protein synthesis itself.

704. Tumor Cell Surface Display of the Immunoglobulin Heavy Chain Fc by Gene Transfer as a Means to Mimic Antibody Therapy

We hypothesized that inducing display of the IgFc molecule on the tumor cell surface by gene transfer would promote tumor cell killing by the same mechanisms as antibody based approaches, but would alleviate some of the problems inherent in the use of antibodies for cancer therapy. We expressed the cDNA of the Fc portion of the murine IgG2a heavy chain on the surface of tumor cells thereby mimicking a natural antibody tagging event. In vitro, Fc receptor positive natural killer cells specifically recognized and lysed B16 melanoma cells expressing surface IgFc. Macrophages bound to B16-Fc, cells significantly more than to parental B16 and surface IgFc expression promoted formation of the terminal complement pore complex. Expression of the IgFc dramatically delayed the ability of B16 cells to form tumors in vivo, dependent on macrophages and NK cells.