Abstract
The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.
Highlights
Membrane proteins represent one of the main pharmaceutical targets
We demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM)
The situation is even more complicated in the case of interfacial membrane protein loops, because the desolvation energy depends on the dielectric permittivity ε that rapidly increases from 2 inside the membrane to 80 in the bulk. ε is likely to be around 10−20 immediately adjacent to the membrane surface,[4] but depending on the exact position of the loop ε may adopt any value between ∼4 and 40.5 Likewise, the uncertainties in both ε and orientation render energy assessments impossible for (i) induced dipole−induced dipole interactions that make a significant contribution to van der Waals forces and (ii) for hydrogen bonds as their force is determined by the distance dependence of charge-dipole interactions
Summary
Membrane proteins represent one of the main pharmaceutical targets. Yet docking studies are fraught with the difficulties of predicting the flexibility of extracellular loops. We demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM).
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