Bogar, F.; Roman Quintero, M.; Horvath, J.; Nasztor, Z.; Mero, M.; Krekic, S.; Veber, A.; Puskar, L.; Der, A.; Heiner, Z.: PAS-Domain Protein Orientation at a Polyelectrolyte Surface Revealed by Infrared Nanospectroscopy, Chiral Vibrational Spectroscopy, and Molecular Dynamics Simulations. Biomacromolecules 27 (2026), p. 3569-3581
10.1021/acs.biomac.5c02743
Open Accesn Version
Abstract:
PAS domains mediate protein–protein interactions that enable functions such as sensing, signaling, dimerization, and localization. The photoactive yellow protein (PYP) from Halorhodospira halophila is a model PAS-domain protein involved in negative phototaxis, yet its signaling partner remains unidentified. Here, we present a method to resolve protein orientations in PAS-domain signaling by combining nano-FTIR and chiral vibrational sum-frequency generation (VSFG) spectroscopy with molecular dynamics simulations and VSFG spectral calculations. As a demonstration, we used a charged homopolypeptide, poly-l-lysine (PLL), as a surrogate binding surface to probe PYP docking. We found that PYP adopts a preferred interfacial orientation driven primarily by dipole–dipole interactions, despite its water-soluble, i.e., cytoplasmic nature. Remarkably, the inferred interaction surface and orientation closely match those observed in PYP homodimers and in a CNBh-PAS heterodimer. This methodology enables in situ determination of protein orientational preferences during protein–protein interactions and may facilitate identification of binding partners in PAS-domain signaling pathways.