Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases.

Correct identification of translational start sites is important for understanding protein function and transcriptional regulation. The annotated translational start sites contained in genome databases are often predicted using bioinformatics and are rarely verified experimentally, and so are not all accurate. Therefore, we devised a simple approach for determining translational start sites using a combination of epitope tagging and frameshift mutagenesis. This assay was used to determine the start sites of three Mycobacterium tuberculosis proteins: LexA, SigC and Rv1955. We were able to show that proteins may begin before or after the predicted site. We also found that a small, non-annotated open reading frame upstream of Rv1955 was expressed as a protein, which we have designated Rv1954A. This approach is readily applicable to any bacterial species for which plasmid transformation can be achieved.

Summary The pathogen Mycobacterium tuberculosis expresses two chaperonins, one (Cpn60.1) dispensable and one (Cpn60.2) essential. These proteins have been reported not to form oligomers despite the fact that oligomerization of chaperonins is regarded as essential for their function. We show here that the Cpn60.2 homologue from Mycobacterium smegmatis also fails to oligomerize under standard conditions. However, we also show that the Cpn60.2 proteins from both organisms can replace the essential groEL gene of Escherichia coli, and that they can function with E. coli GroES cochaperonin, as well as with their cognate cochaperonin proteins, strongly implying that they form oligomers in vivo. We show that the Cpn60.1 proteins, but not the Cpn60.2 proteins, can complement for loss of the M. smegmatis cpn60.1 gene. We investigated the oligomerization of the Cpn60.2 proteins using analytical ultracentrifugation and mass spectroscopy. Both form monomers under standard conditions, but they form higher order oligomers in the presence of kosmotropes and ADP or ATP. Under these conditions, their ATPase activity is significantly enhanced. We conclude that the essential mycobacterial chaperonins, while unstable compared to many other bacterial chaperonins, do act as oligomers in vivo, and that there has been specialization of function of the mycobacterial chaperonins following gene duplication.

Mycobacterium smegmatis contains three chaperonin (cpn60) genes homologous to the Escherichia coli groEL gene. One of these (cpn60.1) is required for biofilm formation, but is nonessential, whereas a second (cpn60.2) is essential. Mycobacterium smegmatis is unique among Mycobacteria in having a third chaperonin gene, cpn60.3. The cpn60.1 gene has a gene upstream (cpn10) that is homologous to the gene for the E. coli co-chaperonin GroES. Phylogenetic analysis of the mycobacterial homologues suggests that early gene duplication and sequence divergence gave rise to the cpn60.1 and cpn60.2 genes found in all Mycobacteria species, while cpn60.3 appears to have been acquired by horizontal gene transfer. Here, we show that cpn60.2 and cpn10 are expressed more strongly than cpn60.1, while cpn60.3 shows very low levels of expression. The expression of all the genes, except cpn60.3, is significantly induced by heat shock, but much less so by other stresses. We mapped mRNA 5'-ends for the cpn10 and cpn60.1 genes, and measured the promoter activity of the upstream regions of both genes. The results show that the mRNA for this operon is cleaved between the cpn10 and cpn60.1 genes. These results are consistent with the evolution of a distinct function for the cpn60.1 gene.