PAS-Domain Protein Orientation at a Polyelectrolyte Surface Revealed by Infrared Nanospectroscopy, Chiral Vibrational Spectroscopy, and Molecular Dynamics Simulations

Bogár, Ferenc and Quintero, Montserrat Roman and Horváth, János and Násztor, Zoltán and Mero, Mark and Krekic, Szilvia and Veber, Alexander and Puskar, Ljiljana and Dér, András and Heiner, Zsuzsanna (2026) PAS-Domain Protein Orientation at a Polyelectrolyte Surface Revealed by Infrared Nanospectroscopy, Chiral Vibrational Spectroscopy, and Molecular Dynamics Simulations. BIOMACROMOLECULES, 27 (6). pp. 3569-3581. ISSN 1525-7797

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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.

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