Fig 1.
Schematic representation of full adhesins MpAFP and MhLap and corresponding SMFS constructs.
The mechanical stability of region II of the full adhesin MpAFP (1.5 MDa) and MhLap (0.3 MDa) (A) is investigated using octameric constructs (B). Region II of MpAFP consists of 120 identical 104-amino-acid repeats, whereas region II of MhLap consists of 25 repeats of 97 amino acids, with on average 76% sequence identity between subsequent repeats. MpAFP RII8-GFP consists of eight MpAFP RII repeats separated into two sections of tetra-tandemers by a GFP protein included in the middle which serves as internal force calibration standard. MhLap RII8 consists of repeats 2–5 and 21–24. Both constructs have two C-terminal cysteines to promote the interaction of the proteins with the gold surface. The full amino acid sequences of the protein constructs are given in Section A in S1 Supporting Information. The pickup of the adhesin construct MpAFP RII8-GFP by the AFM tip is shown schematically in (C).
Fig 2.
Typical sawtooth-like force-extension curves.
Force curves of (A) MpAFP RII8-GFP, (B) MhLap RII8 and (C) I27RS8 were obtained at a pulling speed of 1 μm/s and 10 mM Ca2+. The worm-like chain (WLC) model was applied to analyze the observed unfolding peaks from which we obtain values for a contour length increase ΔLc upon unfolding and a persistence length Lp. The force-distance curve of the MpAFP RII8-GFP displays seven peaks corresponding to the unfolding of RII monomers (red WLC fit) and a much smaller peak at a small extension corresponding to GFP unfolding (green WLC fit). At a 1 μm/s pulling speed we obtained ΔLc = 33.2 ± 3 nm, ΔLc = 33.6 ± 5 nm and ΔLc = 27.3 ± 5 nm for MpAFP RII, MhLap RII and I27, respectively. Experimental data are shown in black; red and green solid lines correspond to WLC fits.
Fig 3.
Pulling speed dependence of MpAFP RII and unfolding force histograms of MpAFP RII, MhLap RII and I27.
(A) A linear dependence of MpAFP RII unfolding force with pulling speed is visible. The measured unfolding forces for I27 at 1 μm/s pulling speed were in agreement with data of I27 taken from Brockwell et al. [32], Carrion-Vazquez et al. [5] and Fowler et al. [33]. (B) Normalized histograms of measured unfolding forces of I27 (N = 349), MhLap RII (N = 1006) and MpAFP RII (N = 518) at 1 μm/s pulling speed.
Fig 4.
MpAFP RII unfolding force depends on Ca2+ concentration.
(A) Overlay of force curves of MpAFP RII8-GFP at 10 mM and 30 μM Ca2+. Force peaks of MpAFP RII are on average ~100 pN lower when the protein is in 30 μM calcium compared to the force peaks of MpAFP RII in 10 mM calcium. Force peak of GFP is unaffected, which is as expected since no Ca2+ ions are bound to GFP. (B) Overlay of four force curves of MpAFP RII8-GFP in 10 mM Ca2+ (left) and 30 μM Ca2+ (right). A larger spread in unfolding force peaks is observed for MpAFP RII at 30 μM Ca2+ compared to MpAFP RII in 10 mM Ca2+. All force measurements were performed at 1 μm/s pulling speed.
Fig 5.
Coordinated calcium ions clamp both MpAFP RII and MhLap RII extender domains.
(A) Amino acid sequence alignment of the MpAFP RII extender domain with eight MhLap RII repeats used in the octa-tandemer construct. Residues highlighted in green coordinate the calcium ions A and B that are involved in clamping. Residues involved in hydrogen bonding of terminal strands are indicated in red. (B) Crystal structure of the third repeat of MpAFP RII tetra-tandemer (4P99) and (C) Phyre2 model of MhLap RII repeat 3. Calcium ions involved in clamping are labelled A and B. Interactions of MpAFP RII residues with Ca2+ ions are indicated with green dashed lines. Hydrogen bonds are indicated with red dashed lines (see Section D in S1 Supporting Information for details).