Faculty Bibliography

The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The beta-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.

Outer surface protein A from the Lyme disease spirochete Borrelia burgdorferi contains a single-layer beta-sheet connecting the N- and C-terminal globular domains. The central beta-sheet consists largely of polar amino acids and is solvent-exposed on both faces, which so far appears to be unique among known protein structures. We show that the single-layer beta-sheet segment is surprisingly stable (deltaG for hydrogen exchange is approximately 8 kcal mol(-1) at 45 degrees C). Possible factors contributing to the stability of the single-layer beta-sheet are discussed based on an analysis of the crystal structure.

Sporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system. Mutants in opp sporulate poorly but second-site revertants can be found that restore sporulation and peptide transport. These second-site mutations were found in a second oligopeptide transport system, app, in which the peptide-binding protein, AppA, is mutant owing to a frame-shift mutation, and the revertants restore the original frame. The AppA mutation is present in the 168 strain of B. subtilis. The app operon consists of five genes in the order appD-appF-appA-appB-appC, with the locus designations corresponding to their homologue in the opp operon. Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA. Both the App and Opp permease systems can transport tetra- and pentapeptides, but tripeptides are not transported by the App system. Strains of the genotype app+ opp- are resistant to the tripeptide antibiotic bialaphos. The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation. The phenotypes raised some speculation about the subunit configuration of the Opp system.

Alkaline phosphatase (APase) is induced as a culture enters stationary phase because of limiting phosphate. The results presented here show that expression of APase is regulated both negatively and positively. PhoP, a homolog of a family of bacterial transcription factors, and PhoR, a homolog of bacterial histidine protein kinases, are required for induction of APases when phosphate becomes limiting. The induction period lasts 2 to 3 h, after which the rate of APase accumulation is decreased. Mutant strains defective in the Spo0A transcription factor failed to decrease APase production. The consequent hyperinduction of APase in a spo0A strain was dependent on phoP and phoR. spo0B and spo0F strains also overexpressed APase, suggesting that phosphorylated Spo0A is required for repression of APase. An abrB mutant allele in the presence of the mutant spo0A allele in these strains did not significantly change the APase hyperinduction phenotype, demonstrating that Spo0A repression of abrB expression is not the mechanism by which Spo0A-P regulates APase expression. Our previous report that spo0A mutants do not express APases is in conflict with the present data. We show here that the previously used mutants and a number of commonly used spo0 strains, all of which have an APase deficiency phenotype, contain a previously unrecognized mutation in phoR.