Dominant Folding Pathways of a WW Domain

We investigate the folding mechanism of the WW domain Fip35 using a realistic atomistic force field by applying the Dominant Reaction Pathways (DRP) approach. We find evidence for the existence of two folding pathways, which differ by the order of formation of the two hairpins. This result is consistent with the analysis of the experimental data on the folding kinetics of WW domains and with the results obtained from large-scale molecular dynamics (MD) simulations of this system. Free-energy calculations performed in two coarse-grained models support the robustness of our results and suggest that the qualitative structure of the dominant paths are mostly shaped by the native interactions. Computing a folding trajectory in atomistic detail only required about one hour on 48 CPU's. The gain in computational efficiency opens the door to a systematic investigation of the folding pathways of a large number of globular proteins

Disposition of Federally Owned Surpluses

PDZ domains are scaffolding modules in protein-protein interactions that mediate numerous physiological functions by interacting canonically with the C-terminus or non-canonically with an internal motif of protein ligands. A conserved carboxylate-binding site in the PDZ domain facilitates binding via backbone hydrogen bonds; however, little is known about the role of these hydrogen bonds due to experimental challenges with backbone mutations. Here we address this interaction by generating semisynthetic PDZ domains containing backbone amide-to-ester mutations and evaluating the importance of individual hydrogen bonds for ligand binding. We observe substantial and differential effects upon amide-to-ester mutation in PDZ2 of postsynaptic density protein 95 and other PDZ domains, suggesting that hydrogen bonding at the carboxylate-binding site contributes to both affinity and selectivity. In particular, the hydrogen-bonding pattern is surprisingly different between the non-canonical and canonical interaction. Our data provide a detailed understanding of the role of hydrogen bonds in protein-protein interactions

Antibody domain exchange is an immunological solution to carbohydrate cluster recognition.

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces

Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture

The transthyretin (TTR) amyloidoses are human diseases in which the misfolded TTR protein aggregates in tissues with subsequent visceral, peripheral, and autonomic nerve dysfunction. Recent reports have stressed the importance of oligomeric intermediates as major cytotoxic species in various forms of amyloidogenesis. We have examined the cytotoxic effects of several quaternary structural states of wild-type and variant TTR proteins on cells of neural lineage. TTR amyloid fibrils and soluble aggregates >100 kDa were not toxic. Incubation of TTR under the conditions of the cell assay and analysis by size-exclusion chromatography and SDS/PAGE reveal that monomeric TTR or relatively small, rapidly formed aggregates of a maximum size of six subunits were the major cytotoxic species. Small molecules that stabilize the native tetrameric state were shown to prevent toxicity. The studies are consistent with a model in which the misfolded TTR monomer rapidly aggregates to form transient low molecular mass assemblies (<100 kDa) that are highly cytotoxic in tissue culture

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association