Topographical and mechanical properties of liposome surfaces harboring Na,K-ATPase by means of atomic force microscopy

In this study, we obtained unprecedented AFM images of the Na,K-ATPase (NKA) pump after being reconstituted into DPPC and DPPC:DPPE liposomes. The mechanical properties observed in the phase images were associated with protrusions correlated to NKA microdomains, which are the darker areas seen in the AFM phase images. Protrusions in the DPPC-NKA proteoliposomes ranged from 38 to 115 nm, with 74 ± 21 nm diameter and 2.1 ± 1.4 nm height. DPPC:DPPE-NKA proteoliposomes showed protrusions from 21 to 78 nm, with 38 ± 16 nm diameter and 0.7 ± 0.5 nm height. We have estimated the presence of annular lipids in the microdomains considering that the areas of the protrusions should contain αβ oligomers and annular phospholipids. For DPPC-NKA proteoliposomes, we hypothesize that 40 phospholipids surround an (αβ)2 dimer and 46 phospholipids are present for the DPPC:DPPE-NKA proteoliposomes in an αβ monomer. Catalytic activity measurements of both lipid compositions of proteoliposomes harboring NKA provide strong evidence regarding the protein orientation in the biomembrane. AFM data suggest that DPPC-NKA proteoliposomes are also rightside-out protein orientated, where the protrusions have an average height of 2.1 nm, while for DPPC:DPPE-NKA proteoliposomes, the majority of the protein reconstituted should be inside-out orientated, where the protrusions' average height is 0.5 nm. This result corroborates with the enzymatic analysis, where 61% and 91% of the enzymatic activity was recovered, respectively. Thus, a new application of AFM as a tool for the determination of topological features of protrusions in proteoliposomes has been brought to the scientific community, in addition to revealing the distinct catalytic orientation of enzymes present in the biomembranes model.