Figure 1.
Construction and binding of MTH719 PMB-GFP fusion variants.
(a) Schematic view of the 574-residue S-layer protein MTH719 with predicted PMB domains. (▾) and (♦) represent the major trypsin and chymotrypsin cleavage sites, respectively, as predicted by Peptide cutter (ExPASy). Arrows labeled 1P, 2P and 3P show the regions used for the GFP fusion proteins. (b) Molecular architecture of different PMB fusion constructs. Numbers indicate the different PMB motifs (see Fig. 1a). GFP and H represent Green Fluorescent Protein and the Histidine10 tag, respectively. At the right, cell wall binding activity is denoted qualitatively.
Figure 2.
Analytical size exclusion chromatography analysis of the PMB domain 3P-His10.
Elution profile using a Superdex 200 10/300 GL size exclusion column of Ni-NTA purified 3P-His10, eluted with 50 mM NaH2PO4, 150 mM NaCl, pH 7.0. Inset shows the calibration plot obtained by column calibration with standard globular proteins ranging in size from 43 kDa to 669 kDa. (*) and (▴) indicates 3P-His10 at pH 7.0 and at pH 9.0 respectively.
Figure 3.
Chemical denaturation of the 3P-His10 PMB domain.
Tryptophan fluorescence of guanidine hydrochloride (GdHCl)-treated 3P-His10 (3 µM in 50 mM NaH2PO4, pH 8.5) at 25°C. The relative increase of the fluorescence intensity (in arbitrary units, a.u) is shown as a function of the concentration of GdHCl, as indicated in the inset.
Figure 4.
Partial proteolysis of the purified 3P-His10, PMB domain.
Coomassie brilliant blue-stained SDS-20% PAA gel containing samples of 3P-His10 PMB in 50 mM NaH2PO4, pH 8.5, digested with 4.5 and 45 µM chymotrypsin and trypsin, respectively. Arrows indicate the major digestion products. Lane 1, low range molecular mass marker; lanes 2, 3 and 5, 6 contain chymotrypsin- and trypsin-liberated fragments, respectively; lane 4 contains undigested 3P-His10 protein.
Figure 5.
PMB domain binding to pseudomurein of methanogenic archaeal cells and bacterial spheroplasts.
(a) Phase contrast and (b) fluorescence microscopy view of 3P-GFP-His10 bound to pseudomurein of Methanobacterium sp. (long rods can still be seen in the cell extract) and L. lactis spheroplasts (ovoid). (c) Phase contrast and (d) fluorescence microscopy view of 2P-GFP-His10, bound only to L. lactis spheroplasts, in a mixture with pseudomurein of Methanobacterium sp. cells (long rods). (e) Phase contrast and (f) fluorescence microscopy view of the PMB domain of PeiW fused with GFP-His6 bound to L. lactis spheroplasts.
Figure 6.
Binding of 3P-GFP-His10 to pseudomurein of methanogenic archaeal cells at different pH conditions.
(a) Phase contrast and (b) fluorescence microscopy views of 3P-GFP-His10 bound to pseudomurein of Methanobacterium sp. cells (long rods) at pH 6.5. (c) Phase contrast and (d) fluorescence microscopy views of 3P-GFP-His10 bound to pseudomurein of Methanobacterium sp. cells at pH 9.0.