KL₄ peptide induces reversible collapse structures on multiple length scales in model lung surfactant

Abstract

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).

Figure 1

(A) Isotherms of a DPPC monolayer during repeated compression (3 curves to the right) and expansion (3 curves to the left) at 25°C. FM image below shows a highly condensed (dark) film at high pressure that eventually undergoes a fracture-like collapse at maximum compression (image from 1st cycle). (B) Isotherms of a POPG monolayer during repeated compression and expansion at 25°C. FM image below shows a fluid (bright) film with no condensed domains that undergoes vesicular collapse at maximum compression (image from 1st cycle). (C) Isotherms of a DPPC/POPG (7:3) monolayer during repeated compression and expansion at 25°C. FM image below shows the folding collapse at maximum compression (image from 1st cycle).

Figure 2

(A) Isotherms of a DPPC/POPG (7:3) monolayer with 8 wt% KL₄ during repeated compression (3 curves to the right) and expansion (3 curves to the left) at 25°C. FM image below shows the change in folding collapse at maximum compression induced by KL₄ (image from 2nd cycle). (B) Isotherms of a POPG monolayer with 16 wt% KL₄ during repeated compression and expansion at 25°C. FM image below shows the microsilo collapse at maximum compression (image from 1st cycle). (C) Isotherms of a DPPC monolayer with 16 wt% KL₄ during repeated compression and expansion at 25°C. FM image below shows the suppressed folding collapse at maximum compression induced by KL₄ (image from 1st cycle). (D) Series of FM images from the 2nd cycle showing the reincorporation of folds upon expansion of a DPPC/POPG (7:3) monolayer with 8 wt% KL₄. The surface pressure (π) during expansion is indicated in each image. (E) Series of FM images from the 2nd cycle showing the complete reincorporation of microsilos upon expansion of a POPG monolayer with 16 wt% KL₄. The surface pressure (π) during expansion is indicated in each image.

Figure 3

(A) Isotherms of a DPPC/POPG (7:3) monolayer with 4, 8, and 16 wt% KL₄ during compression at 25°C. Dashed line indicates pressure of monolayers inspected by FM and by AFM after deposition onto solid mica substrates. (B) Isotherms of a DPPC/POPG (7:3) monolayer with 16 wt% KL₄ during repeated cycles of compression (3 curves to the right) and expansion (3 curves to the left) at 25°C. (C) FM images of DPPC/POPG (7:3) monolayers with 0, 4, 8, and 16 wt% KL₄ at π ∼50 mN/m. The dark regions are the DPPC-rich condensed domains and the bright regions the POPG-rich fluid matrix. With 16 wt% of KL₄, condensation is no longer visible in FM. (D and E) AFM images at 50 × 50 μm (D) and 5 × 5 μm (E) scan size of DPPC/POPG (7:3) monolayers with 0, 4, 8, and 16 wt% KL₄ deposited onto solid mica substrates at π ∼50 mN/m in a single compression experiment, showing an increase in number density, diameter, and height of nanosilos (bright structures). In AFM images of monolayers without peptide the condensed domains are brighter due to an increased height ∼1 nm above the fluid matrix. Upon addition of KL₄, the condensed phases become hard to discern in AFM of monolayers based on lipid monolayer height contrast, due to the large height of the peptide-induced nanosilos in the fluid matrix. Accordingly, the location of condensed domains is revealed by areas where nanosilos are absent (see for example voids in nanosilo distribution in 8 wt% KL₄). (F) Section analysis of individual nanosilo indicated with box in E.

Figure 4

(A) Overlay of isotherms from one cycle of compression and expansion of POPG + 16 wt% KL₄ and DPPC/POPG (7:3) monolayers with a DPPC/POPG (7:3) + 16 wt% KL₄ monolayer support a mechanistic connection between the nano- and microscale material transformations taking place during compression of model lung surfactant (see text for details). Inset shows the increase in amplitude of π oscillations after addition of KL₄ during final collapse of the surfactant film induced by the increased fold size as shown in FM images in B.