PAS-Domain Protein Orientation at a Polyelectrolyte Surface Revealed by Infrared Nanospectroscopy, Chiral Vibrational...

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.