Macrophages orchestrate the immune response to tumor cell death.

The mechanisms by which the immune system distinguishes normal developmental cell death from pathological immunogenic cell killing are central to effective cancer immunotherapy. Using HSVtk suicide gene therapy, we showed that macrophages can distinguish between tumor cells dying through classical apoptosis and tumor cells engineered to die through nonapoptotic mechanisms, resulting in secretion of either immunosuppressive cytokines (interleukin 10 and transforming growth factor beta) or inflammatory cytokines (tumor necrosis factor alpha or interleukin 1beta), respectively. Additionally heat shock protein 70 acts as one component of a bimodal alarm signal that activates macrophages in the presence of stressful, immunogenic tumor cell killing. These differential responses of macrophages can also be used to vaccinate mice against tumor challenge, using adoptive transfer, as well as to cure mice of established tumors.

Long-range order in the src SH3 folding transition state.

One of the outstanding questions in protein folding concerns the degree of heterogeneity in the folding transition state ensemble: does a protein fold via a large multitude of diverse "pathways," or are the elements of native structure assembled in a well defined order? Herein, we build on previous point mutagenesis studies of the src SH3 by directly investigating the association of structural elements and the loss of backbone conformational entropy during folding. Double-mutant analysis of polar residues in the distal beta-hairpin and the diverging turn indicates that the hydrogen bond network between these elements is largely formed in the folding transition state. A 10-glycine insertion in the n-src loop (which connects the distal hairpin and the diverging turn) and a disulfide crosslink at the base of the distal beta-hairpin exclusively affect the folding rate, showing that these structural elements are nearly as ordered in the folding transition state as in the native state. In contrast, crosslinking the base of the RT loop or the N and C termini dramatically slows down the unfolding rate, suggesting that dissociation of the termini and opening of the RT loop precede the rate-limiting step in unfolding. Taken together, these results suggest that essentially all conformations in the folding transition state ensemble have the central three-stranded beta-sheet formed, indicating that, for the src homology 3 domain, there is a discrete order to structure assembly during folding.

Evolutionary conservation in protein folding kinetics.

The sequence and structural conservation of folding transition states have been predicted on theoretical grounds. Using homologous sequence alignments of proteins previously characterized via coupled mutagenesis/kinetics studies, we tested these predictions experimentally. Only one of the six appropriately characterized proteins exhibits a statistically significant correlation between residues' roles in transition state structure and their evolutionary conservation. However, a significant correlation is observed between the contributions of individual sequence positions to the transition state structure across a set of homologous proteins. Thus the structure of the folding transition state ensemble appears to be more highly conserved than the specific interactions that stabilize it.

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 beta-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 beta-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.

Functional rapidly folding proteins from simplified amino acid sequences.

Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure.

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.