Category:PurpleSU18

This study about PspC topology allows authors to propose a new working model for activation of the Psp system. Although there have been conflicting past studies on the topology of the shock protein PspC, through multiple direct assays and an in silico analysis with TOPCON, the server for membrane protein topology prediction, the study shows that both the N and C termini of the protein are located in the cytoplasm. The C terminus, which might be the recognition site for FtsH protease and an interaction interface with PspA, would function in the cytoplasm, not the periplasm. In the assay with chemical modification of cysteine residues, seen in Figure 4, CpxP is shown to be located in the periplasm and confirms that 3-maleimidylpropionyl biocytin (MPB) can cross the outer membrane, as in Figure 4A. All PspB cysteine substitution mutants were labeled by MPB only after cell disruption, agreeing with its C terminus location in the cytoplasm, as in Figure B, which is information that has been supported well before. However, all of the PspC cysteine substitution mutants were also labeled only after sonication, whether the cysteine was in the N or C terminus or not, which is shown by the image 4C. This confirms that both ends of PspC are on the cytoplasmic side of the membrane.

Bacteriophage T4 segB gene encodes for a protein of unknown function, but is homologous to homing endonucleases of the GIY-YIG family. By using sequence alignment with PCR, the study demonstrates that the SegB protein is a site-specific endonuclease, by analyszing the DNA cleavage site. The cleavage site analysis suggests SegB is a homing endonuclease that functions to spread its own gene among T4-related phages. In Figure 3, it is shown that Phage T4 SegB protein demonstrates endonuclease activity by cleaving DNA in the presence of Mg2+, Mn2+ or Ca2+ cations. Although the study uses a wet lab, the focus is on sequencing; the evidence is sequence alignment, instead of direct assay.