The predicted amino acid sequence of Pam gives little clue to its role or of the potential structure that mediates its adhesive properties. To get an insight into the structure of Pam, we analyzed the protein with circular dichroism spectroscopy. Our far-UV CD data HKI272 strongly indicate that Pam is a helical protein, with 5.5

helix segments per 100 residues and an average helix length of 10.5 residues. By contrast, only 8% of residues are expected to Epigenetics inhibitor form β-strands. We obtained only very weak spectra for Pam in the near-UV wavelengths, but 1D 1H and 2D 1H-15N HSQC NMR spectra (data not shown) and high melting temperature from differential scanning calorimetry experiments confirm that the protein has well defined tertiary structure. A degree of tertiary structural prediction is available from the far-UV spectra, specifically the position of the spectral cross-over from positive to negative, and the magnitude of the negative maximum at 208 nm [20]. These both suggest that Pam is a α+β protein. Rather than having intermixed segments, such proteins have separate α-helix and β-sheet-rich regions [21]. Interestingly, although Pam is not secreted at 37°C in P. asymbiotica, it shows thermal buy CB-839 stability far beyond this.

Differential scanning calorimetry revealed that the protein does not begin to thermally denature until heated to temperatures above 60°C. The transition midpoint is 77.4°C, suggesting that Pam is particularly thermostable for a protein produced by an organism considered to be psychrophilic [22]. In fact, this midpoint is approaching that seen in thermophilic bacteria and archaea [23–25]. Without high resolution structural analyses we are unable to explore precise contributions to the thermal stability of Pam, but the high

α-helix content is likely to be significant; thermostable proteins are richer in α-helices than mesophilic proteins [26]. The observed ability IKBKE of Pam to refold to its native conformation following denaturation may be biologically significant; this folding indicates that the protein can form its native structure in the absence of molecular chaperones, outside of the cell if it is secreted as an unfolded polypeptide. It is as yet not clear how Pam is secreted from the cell as we can detect no recognizable signal motifs, neither were found in Pit [10]. Finally, although the role of this highly secreted protein in Photorhabdus biology has not yet been completely elucidated, we have shown its possible relevance in cell attachment. Our findings indicate that Pam is a secreted adhesive factor of Photorhabdus that modifies attachment of cells to surfaces in biotic (hemolymp) and abiotic (SPR) conditions.