Optical Control of the Folding and Membrane Topology od Helical Transmembrane Protein Fragments

Laburpena

The spontaneous (translocon-unassisted) folding/insertion of helical transmembrane (TM) protein fragments into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion [1]. Understanding the molecular mechanism underlaying these steps has been challenged by the lack of suitable experimental approaches to perturb protein fragments rapidly and reversibly out of equilibrium once reconstituted in membranes. We have been actively working in implementing a strategy to control/perturb the folding and membrane topology of protein fragments with light. We have started with a 24-residues-long hydrophobic α-helical polypeptide from the KALP family as a model for a helical transmembrane protein fragment. We introduced two Cys to KALP and coupled it to an azobenzene photoswitch (KCALP-azo), as done before for helical soluble protein fragments [2]. By polarized FTIR spectroscopy we determined that KCALP-azo (trans azobenzene) folds as a TM α-helix, as KALP does, with a helix tilt angle of 32 ± 5. After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), we confirmed the optical control of the membrane topology of KCALP-azo: its helix tilt changes reversibly from 32 ± 5° (TM topology, blue light) to 79 ± 8° (MI topology, UV light) [3].We have started to use short UV and blue light pulses in combination with time-resolved FTIR difference spectroscopy [4]. These experiments exploit the here achieved optical control to provide a detailed picture on how a membrane interface helical fragment inserts in lipidic membrane, a relevant information currently only available from MD simulations [5].