Faculty Bibliography

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Angstroms resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel beta-sheet. These well-defined water molecules provide additional structural links between adjacent beta-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA.

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.

The estrogen receptor-alpha is a wonderfully complex protein important in normal biology, breast cancer, and as a target for anti-cancer agents. We are using the available structures of the hERalpha as well as secondary structure predictions to guide site-directed mutagenesis in order to test the importance of specific interactions and regions in the ligand-regulated activity of the protein. In one area of interest, we are investigating the role of the F domain in the ligand-stimulated activity of the hERalpha. Results from our laboratory and others suggest that the F domain modulates the activity of the hERalpha. In order to better understand the role of the F domain in the hERalpha, we have constructed mutants within this region. Mutations within a predicted alpha-helical region alter the response of the ER to estradiol (E2), eliminate or impair the agonist activity of 4-hydroxytamoxifen (4-OHT), and alter the ability of E2 to overcome 4-OHT's antagonist activity. Deleting the F domain increases the affinity of the receptor for E2; by contrast, mutating a residue in the middle of the predicted helix to a proline does not alter the affinity for E2, but does change the binding mechanism from a positive cooperative to a noncooperative interaction. These and other results show the F domain exhibits substantial functional complexity, and support the idea that this domain modulates the activity of the hERalpha. In a second area of interest, we are investigating the role of hydrophobic and hydrogen-bonding interactions at the start of helix 12 in the activity of the hERalpha. Leucine-536 (L536) has been proposed to participate in hydrophobic interactions that form part of a capping motif stabilizing the start of helix 12. When mutated, the resulting receptors exhibit a reduced response, or even an inverted response, to E2 and 4-OHT on both ERE-driven and AP-1-driven promoters. Interestingly, these mutated receptors also exhibit altered interactions with probes that recognize the agonist-bound and 4-OHT-bound conformations of the ERalpha. Thus, L536 couples the binding of ligand with the conformation of the receptor. Overall, these results show that combining structure-based hypotheses with functional tests of the ER's activity can identify regions and interactions that are important in the ligand-stimulated activity of the protein.

The tenth fibronectin type III (FN3) domain of human fibronectin (FNfn10), a prototype of the ubiquitous FN3 domain, is a small, monomeric beta-sandwich protein. In this study, we have bisected FNfn10 in each loop to generate a total of six fragment pairs. We found that fragment pairs bisected at multiple loops of FNfn10 show complementation in vivo as tested with a yeast two-hybrid system. The dissociation constant of these fragment pairs determined in vitro were as low as 3 nM, resulting in one of the tightest fragment complementation systems reported so far. Furthermore, we show that the affinity of fragment complementation is correlated with the stability of the uncut parent protein. Exploring this correlation, we screened a yeast two-hybrid library of one fragment and identified mutations that suppress the effect of a destabilizing mutation in the other fragment. One of the identified mutations significantly increased the stability of the uncut wild-type protein, proving that fragment complementation can be used as a novel strategy for the selection of proteins with enhanced stability.

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.

Here, we describe a structure-based approach to reduce the size of an antigen protein for a subunit vaccine. Our method consists of (i) determining the three-dimensional structure of an antigen, (ii) identifying protective epitopes, (iii) generation of an antigen fragment that contains the protective epitope, and (iv) rational design to compensate for destabilization caused by truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine. Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi elicits protective immunity that blocks transmission of Borrelia from the tick vector to the vaccinated animal, and thus has been a focus of vaccine development. OspA has two globular domains that are connected via a unique single-layer beta-sheet. All anti-OspA monoclonal antibodies that block Borrelia transmission bind to conformational epitopes in the C-terminal domain of OspA, suggesting the possibility of using the C-terminal domain alone as a recombinant protein-based vaccine. The removal of ineffective parts from the OspA antigen may reduce side effects and lead to a safer vaccine. We prepared a C-terminal fragment of OspA by removing approximately 45% of residues from the N terminus. Although the fragment retained the native conformation and affinity to a protective antibody, its vaccine efficacy and conformational stability were significantly reduced with respect to full-length OspA. We successfully stabilized the fragment by replacing amino acid residues involved in buried salt-bridges with residues promoting hydrophobic interactions. The mutations promoted the vaccine efficacy of the redesigned fragment to a level comparable to that of the full-length protein, demonstrating the importance of the antigen stability for OspA's vaccine efficacy. Our strategy should be useful for further refining OspA-based vaccines and developing recombinant vaccines for other diseases.

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.

It is challenging to experimentally define an energy landscape for protein folding that comprises multiple partially unfolded states. Experimental results are often ambiguous as to whether a non-native state is conformationally homogeneous. Here, we tested an approach combining systematic mutagenesis and a Bronsted-like analysis to reveal and quantify conformational heterogeneity of folding intermediate states. Using this method, we resolved an otherwise apparently homogeneous equilibrium folding intermediate of Borrelia burgdorferi OspA into two conformationally distinct species and determined their relative populations. Furthermore, we mapped the structural differences between these intermediate species, which are consistent with the non-native species that we previously proposed based on native-state hydrogen exchange studies. When treated as a single state, the intermediate ensemble exhibited fractional Phi-values for mutations and Hammond-type behaviors that are often observed for folding transition states. We found that a change in relative population of the two species within the intermediate ensemble explains these properties well, suggesting that fractional Phi-values and Hammond-type behaviors exhibited by folding intermediates and transition states may arise more often from conformational heterogeneity than from a single partial structure. Our results are consistent with the presence of multiple minima in a rugged energy landscape predicted from theoretical studies. The method described here provides a promising means to probe a complex folding energy landscape.