The active transport of ions across biological membranes requires their hydration shell to interact with the interior of membrane proteins. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Using the octahelical transmembrane architecture of the copper-transporting P1B -type ATPase from Legionella pneumophila as a model structure, we have established the site-specific labeling of internal cysteines with a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the protein and in its lipid-embedded state in nanodiscs. Time-dependent fluorescence shifts revealed the site-specific hydration and dipole mobility around the conserved ion-binding motif. The spatial distribution of both features is shaped significantly and independently of each other by membrane lateral pressure.

Helmholtz Zentrum Dresden Rossendorf Institute of Resource Ecology Bautzner Landstrasse 400 01328 Dresden Germany

Institute for Experimental Physics 1 Universität Leipzig Linnéstrasse 5 04103 Leipzig Germany

J Heyrovský Inst Physical Chemistry of the A S C R v v i Prague Czech Republic

Nationales Zentrum für Tumorerkrankungen Heidelberg Im Neuenheimer Feld 460 69120 Heidelberg Germany

Technische Universität Dresden Biotechnology Center Tatzberg 47 49 01307 Dresden Germany

University of Bern Dept of Clinical Pharmacology Murtenstrasse 35 3008 Bern Switzerland

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What Does Time-Dependent Fluorescence Shift (TDFS) in Biomembranes (and Proteins) Report on?