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. 2021 Feb 8;10:e64302. doi: 10.7554/eLife.64302

Figure 3. GplG.

(a) Construct design, c.f., Figure 2a. The N-terminal LepB fusion is indicated. (b) Force profiles (FPs) for GlpG and LepB-GlpG (N = 131–224) (orange), NTD(F16E) (green), in vitro translated N-terminal domain (NTD) (magenta), and NTD(F16E) (black), LepB-GlpG with SecM(Ec-Sup1) AP (blue), and coarse-grained molecular dynamics (CGMD)-FP calculated with a −100 mV membrane potential (gray). Error bars indicate SEM values. Note that the LepB-GlpG constructs are two residues shorter than the corresponding GlpG constructs but are plotted with the same N values as the latter to facilitate comparison. (c) NTD (PDB ID: 2LEP), with F16 in spacefill. (d) Enlarged FPs for LepB-GlpG with SecM(Ec) AP (orange), SecM(Ec-Ms) AP (green), SecM(Ec-sup1) AP (blue), and GlpG(Y138F139L143→NNN) with SecM(Ec-Ms) AP (magenta). CGMD-FP in gray. (e) Structure of GlpG with the periplasmic surface helix in blue, TMH2 in red, the membrane-associated cytoplasmic segment in cyan, and TMH5 in yellow. Y138F139L143 and G222I223Y224L225 are shown as sticks. (f) LepB-GlpG peak III-a and III-c sequences aligned, respectively, from their Nstart and Nmax values, and the mutant LepB-GlpG(Y138F139L143→NNN) peak III-c sequence aligned from its Nmax value. Hydrophobic transmembrane helix (TMH) segments are shown in orange and transmembrane α-helices (PDB: 2IC8)underlined. The periplasmic surface helix is italicized. AP: arrest peptide; PTC: polypeptide transferase center.

Figure 3.

Figure 3—figure supplement 1. GlpG.

Figure 3—figure supplement 1.

(a) fFL values for peak III obtained for LepB-GlpG fusion constructs (orange) and GlpG constructs calculated either including (dashed green) or excluding (dashed magenta) the slowly migrating band indicated in Figure 1—figure supplement 1j,k in IFL. The two latter are from single measurements. (b) As in Figure 3b, but with a hydrophobicity plot (HP) (ΔG) calculated by TOPCONS (Hessa et al., 2007; Tsirigos et al., 2015) (gray). (c) Sequences corresponding to peaks II–VII aligned based on the Nstart values. The periplasmic surface helix upstream of TMH2 and the hydrophobic patch upstream of TMH5 are in italics. Hydrophobic transmembrane helix (TMH) segments are shown in orange and membrane-embedded α-helices underlined. (d) Sequences corresponding to peaks II–VII aligned based on the Nend values. Hydrophobic TMH segments are shown in orange and membrane-embedded α-helices underlined. PTC: polypeptide transferase center.