Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy

The Na⁺-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K⁺ during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K⁺ binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions.     

Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.