# Protein Dynamics in F-like Bacterial Conjugation
## 4 Mating Pair Stabilization Proteins and the Dynamics of their Structural Ensemble
### TraG and the Structural Dynamics Effectuating its Trifunctional Roles  
TraG\({}_{\text{F}}\) is a 102.5 kDa protein with an N-terminal TM domain bound in the IM and a periplasmic CTD from residues Ala452-Glu939 denoted TraG\({}^{\star}\)[79]. It is one of the largest proteins encoded by the _tra_ region of the F plasmid. Frameshift mutations in the NTD of the protein have been shown to effect the polymerization of pilus subunits, while the whole protein must be intact for Mps and entry exclusion (Eex) events to occur [79; 111]. It was previously thought that the presence of a signal peptidase I cleavage site after residue Ala451 releases TraG\({}^{\star}\) into the periplasm to fulfill its role in Mps, however it appears the CTD must be anchored to the IM for proper TraG\({}^{\star}\) function [78; 79]. The mechanism for the assembly of pilin subunits into polymers as aided by the N-terminus of TraG is not completely understood as indicated in Section 3.2.1; mutations in TraE, K, B, V, C, W, U, F, H, and the N-terminal region of TraG all result in accumulation of mature pilin in the IM [78; 99]. In performing Eex, TraG is predicted to scan the neighboring recipient IM to interact with TraS, which would require the protein to interact over two OM layers. This distance can be approximated to 35-40 nm, assuming donor and recipient OMs are proximal after surface exclusion (Sfx) [112]. As this distance is likely unachievable by a single globular protein, the steps of Mps are thought occur first, and the C-terminal region of TraG is theorized to undergo a reversible conformational change and thrust into the mating pore formed after TraN interacts with OmpA of the recipient cell, potentially through interaction with TraN [79]. In this predicted model, TraG maintains an intact structure and extends TraG* through the mating pore into the periplasmic space of the recipient cell to perform its role in Ex. A TraS-TraG complex has not, however, been detected in mating cells through crosslinking or immunoprecipitation experiments, nor interaction detected using a bacterial two-hybrid system [79]. As well, interaction between TraN and TraG has not been detected using similar methods [102]. The likely cause for the lack of detectable assembly is due to the small number of interacting protein partners in a mating pair, as well as the transient nature of the PPIs. There is a region in the aa sequence of TraG\({}_{\text{F}}\) and TraG\({}_{\text{R100}}\) that shows 55.7% similarity while the proteins hold an overall sequence identity of 93% [79]. Residues Thr610-Ala673 of TraG were predicted to be responsible for the trans-exclusive interaction between TraG and a cognate TraS in the Eex process, as TraG will not interact with TraS in the same IM. This region has been further specified to Thr606-Asp608 in the IncJ plasmid R391 [113]. In all cases this region is part of the periplasmic CTD, which is further evidence of the role TraG* performs in Ex. The homology between TraS\({}_{\text{F}}\) and TraS\({}_{\text{R100}}\) and the interaction between TraS and TraG are discussed in Section 6.2. There is a lack of structural data for TraG from the F-like family; however, useful information is gleaned from other conjugative systems. The canonical T4 SS of the IncN type plasmids is the Ti pilus from _A. tumefaciens_, largely due to the utility of this system in the genetic modification of plants mediated by the Ti plasmid [114; 115]. pCRY, a similar plasmid from the same family, contains the gene product VirB8, a protein orthologous to TraG*. There is evidence indicating the evolution of the _traG_ gene in the F plasmid involved the fusing of an ancestral homolog of genes _virB6_ and _virB8_ to produce a single gene [55]. The structure of VirB8 has been solved in numerous species; _A. tumefaciens_, _Brucella suis_, _Bartonella quintana_, _Bartonella tribocorum_, and _Rickettsia typhi_[116; 117; 118]. As well, TraH, a VirB8 homolog from the plasmid pIP501 (from IncN family, similar to pIPO2T) in _Enterococcus faecalis_ was solved [119].