EPR environmental parameters reveal conformational differences in the membrane-embedded Na_VSp1 and Na_VSp1-P. (A) A plot of residue environmental parameters for Na_VSp1-P (shown in gray) and Na_VSp1 (shown in color). ΔH_o⁻¹ parameter (top); O₂ accessibility, ΠO₂ (middle); water accessibility, ΠNiEDDA (bottom). The EPR parameters for continuous positions are linked by solid lines. Residues that were not studied (because of sub-optimal expression/oligomeric stability) appear as gaps. Positions revealing prominent changes are highlighted within blue circles. (B) Difference in ΠO₂ values between the Na_VSp1 and Na_VSp1-P are mapped on the Na_VAb structure (PDB ID 3RVY) and color-coded, red denoting an increase and blue representing a decrease in the environmental parameter. (C) A plot of the average lipid accessibility (ΠO₂) as a function of the average mobility parameter ΔH_o⁻¹ for the turret loop (yellow), P1 helix (green), and P2 helix (magenta). The light gray boxes represent Na_VSp1-P, and the black boxes denote Na_VSp1. The dotted lines connecting the light and black boxes for each region signify the magnitude of difference between the EPR environmental parameters in the two channels.