# Protein Dynamics in F-like Bacterial Conjugation
## 6 Exclusion Proteins and their Low Structural Dynamicity
### TraS  
TraS is a small, highly hydrophobic IM protein which functions solely in Eex through interaction with TraG, no other TraS interactions have been proposed to occur [79; 81]. The 149 aa protein has not been studied thoroughly as it is difficult to express and purify; the only hydrophilic domain is from residues Glu10-Arg24 [79; 155]. Unlike TraT, TraS does not appear to have a signal sequence and evidently is not a lipoprotein [156]. In further comparing the protein to TraT, sequence conservation between different F-like plasmids is much lower. TraS\({}_{\text{F}}\) and TraS\({}_{\text{R100}}\) have the highest sequence identity at 17%, and where TraT\({}_{\text{F}}\) had its lowest protein sequence identity to TraTpED208, TraS\({}_{\text{PED208}}\) has no sequence homology to TraS\({}_{\text{F}}\)[79]. This diversification in protein sequence may serve as the reason forthe 500-1000-fold higher effect of blocking mating by Eex in comparison to the Sfx system [11; 79]. As mentioned previously, Mps has been suggested to involve interaction of TraG in the donor cell with the IM of the recipient, thereby fusing the cells together [11]. This could be interrupted by TraS and therefore be the mechanism by which Eex prevents an essential step in the formation of the conjugative pore for F-like T4 SS.  
As few structural studies on TraS from F-like plasmids have been performed, structural predictions can only be made based on other forms of data. As mentioned, TraS\({}_{\text{F}}\) and TraS\({}_{\text{R100}}\) hold low sequence identity and participate in entry exclusion when conjugative mating assays are performed with chimeras of non-cognate TraG [79]. As chimeras of TraS have not been expressed successfully due to their instability, likely due to membrane insertion issues, no predictions have been made about the region that exhibits TraG-binding specificity. The number of TM domains in TraS\({}_{\text{F}}\) and TraS\({}_{\text{R100}}\) are predicted to be similar (3 or 4); however, their locations in the position of the sequence are significantly different. Another study hypothesized that Eex specificity lies in the C-terminal half of TraS from the R391 plasmid, but this has not been confirmed in TraS from F-like plasmids [113].  
Entering the TraS\({}_{\text{F}}\) sequence into prediction software such as Phyre2 results in similar predictions: four TM helices are present, and the hydrophilic helix in the N-terminal is recognized as it was the only region modeled, but with poor certainty (38% confidence) [20]. The lack of homologs for the TM portion of TraS is indicative of the absence of structural data for small TM proteins in the PDB. The protein is predicted to be highly structured and all \(\alpha\)-helical (90% of total sequence); thus, displaying how a highly dynamic protein TraG interacts with a well-structured protein TraS to perform its function, likely indicating an induced conformation upon the interaction of TraG with TraS in the IM of the recipient cell. The TraG-TraS interaction is a target for manipulation by novel drugs, as recent studies have shown the most notable genetic difference linking plasmid exclusion to the slow development of plasmid-mediated antibiotic resistance was the presence of a functional _traS_-family gene for Eex events [157].