Protein Crystal Structures
Crystal structures of TaMurJ in both inward closed (PDB:6NC6) and outward (PDB:6NC9) states, along with the EcMurJ crystal structure (PDB:6CC4) in an inward closed state, were visualized. Specific regions, including the N-lobe, C-lobe, and TMs 13-14, were highlighted with distinct colors for detailed analysis.
The full construct of the EcMurJ BRIL fusion protein in an inward closed state was also determined. This structure revealed a distortion of TM1 and indicated the presence of deleted C-terminal residues.
Visualization of TaMurJ and EcMurJ crystal structures provided detailed insights into their inward and outward states, with the EcMurJ BRIL fusion protein structure revealing specific conformational features like TM1 distortion and C-terminal deletions.
Experimental Constructs and Functional Assays
Schematics illustrating co-expression constructs for Sgls and MurJ were prepared to facilitate functional studies.
Various EcMurJ constructs were tested in a MurJ-depletion bacterial strain, where genomic EcMurJ expression is controlled by an arabinose promoter. Crucially, in the absence of arabinose, cell viability relied on the expression of a complementing MurJ construct under an IPTG-inducible promoter.
The EcMurJ BRIL construct, which was used for structural studies, demonstrated complementation comparable to wild-type MurJ activity. A non-functional R24A mutant served as a negative control in these experiments, confirming the specificity of the functional assays.
A lysis assay was conducted to compare His-tagged SglM in the absence and presence of co-expressed EcMurJ BRIL. This assay confirmed that EcMurJ BRIL could effectively rescue cell lysis, further validating its functional integrity.
Functional assays in a MurJ-depleted strain confirmed that the EcMurJ BRIL construct used for structural studies effectively complemented wild-type MurJ activity, rescuing cell viability and preventing lysis.
Protein Complex Analysis
Representative size exclusion chromatography profiles and SDS-PAGE analyses were performed on purified Sgl–MurJ and Sgl–MurJ–Fab–Nb complexes. Molecular weight markers were included for reference during these analyses.
The SglCJ3 band was notably not observed on the SDS-PAGE gel. This absence was attributed to low sensitivity, though its predicted location on the gel was noted.
Analysis of Sgl–MurJ and Sgl–MurJ–Fab–Nb complexes using size exclusion chromatography and SDS-PAGE provided insights into their purification and composition, albeit with some limitations in detecting specific low-abundance components.