The last contact involves the binding of the hydrophobic signal peptide (in the case of a secretory protein) or a TM (in the case of a nascent membrane protein) to a so-called lateral gate of SecYEG consisting of two of α- its hydrophobic helices. , TM2a and TM7, (Driessen, 2008; van den Berg, 2004; Plath, 1998). Conformational changes in the GTPase modules of the signal recognition particle and its receptor induce the initiation of protein translocation. Another amphiphilic helix at the N terminus of the A domain also contributes to lipid binding of FtsY (Weiche, 2008).
This probe monitors the final conformational stage of the SRP•FtsY complex, the activated state (Zhang, 2009). In the presence of liposomes, formation of the SRP•FtsY complex in the presence of a GTP analog, 5'-. If this were true, phospholipids should preferentially stabilize the formation of the closed/activated SRP•FtsY complex.
The pre-organization of FtsY also explains the enhancement of the basal GTPase activity of FtsY and the specific stabilization of the SRP•FtsY complex in the closed/activated states ( Fig. 9A, ΔG , black vs. red). Once the cargo is unloaded, GTP hydrolysis drives the disassembly of the SRP•FtsY complex. Equilibrium titration of the activated SRP•FtsY complex in the absence (left) and presence (right) of 2 mM liposomes.
Michaelis-Menten assay of the basal GTPase reaction of FtsY was performed in the presence (red) and absence (black) of 2 mM liposomes.
CHAPTER 2
We asked whether and how SecYEG regulates the conformation of SRP and FtsY GTPases in the RNC•SRP•FtsY targeting complex. To test this hypothesis, we determined the effect of SecYEG on the rate of GTP hydrolysis of the SRP•FtsY (kcat) complex. Role of Sec61α in the regulated transfer of the ribosome-eastern chain complex from the signal recognition particle to the translocation channel.
Demonstration of a multistep mechanism for the assembly of the SRP•SR receptor complex: implications for the catalytic role of SRP RNA. Liposomes do not reactivate the GTPase activity of the targeting complex in the presence of RNC. In the simplest model (Fig 7E, model (i)), binding of SecYEG first results in displacement of the SRP N domain from the ribosome.
In contrast, our assay specifically reports on the engagement of the signal sequence with the SRP M domain when a stable RNC•SRP complex is formed. The reduction in binding affinity arises from significantly weaker interaction of the SRP N domain with the ribosome and possibly a repositioning of the signal sequence in the M domain. This is followed by transfer of the signal sequence from the SRP M domain to SecYEG (step 6).
The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome. Conformational changes in GTPase modules of the signal recognition particle and its initiation of protein translocation. Role of Sec61 alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel.
Demonstration of a multistep mechanism for the assembly of the SRP.SRP receptor complex: implications for the catalytic role of SRP RNA. Conformational changes in the GTPase modules of the signal recognition particle and its initiation of protein translocation.
CHAPTER 4
We monitored their interaction with SecYEG as we increased the length of the mature region to mimic a nascent elongated chain. Interestingly, as the length of the nascent chain increases, the binding affinity of RNC for SecYEG first weakens and then tightens for both nascent chains. DDM is able to discriminate between types of signal sequences and can sense the length of the nascent chain.
The gate acts as an exit site for TMs of the nascent membrane protein that partitions into the lipid bilayer. We show that YidC weakens the affinity of RNC for SecYEG and changes the conformation of the signaling anchor bound to SecYEG. Furthermore, slightly higher FRET values were observed with longer nascent chains (compare 85 aa to 135 aa in Figure 3B), suggesting that the signal peptide of the longest nascent takes on a slightly different orientation, likely dictated by the length of the nascent chain .
One of the surprising aspects of our results is the lack of dependence of the signal peptide conformation in SecYEG on the length of the nascent chains. In the future, it would be interesting to investigate the topology of the signal peptide with even shorter starting chains. We asked whether detergent-solubilized SecYEG in the absence of lipid bilayers or accessory protein factors is able to distinguish between the nascent chains of different lengths.
We observed a weakening and tightening of the affinity of RNC for SecYEG as the length of the nascent chain increased. Conducting these studies with the expanded repertoire of the emerging chain lengths may answer this question in the future. Successful assembly of the nanodisks was confirmed by gel filtration chromatography (Figure 9B), native (Figure 9C), and denaturing gels (Figure 9D) (see Materials and Methods for detailed description of the reconstitution of SecYEG into nanodisks).
Fitting the titration data gave a dissociation constant similar to that obtained with SecYEG in detergent (Figure 10B). Moreover, the effect of the change in lipid vesicle curvature on the insertion cannot be ruled out. We next examined the efficiency of cotranslational insertion of Lep into proteoliposomes composed of E.
