Abstract
PAS domains mediate protein-protein interactions, which give them key functions ranging from sensing and signaling to dimerization and localization. PYP is one of the PAS-domain model proteins that regulates negative phototactic signal response, and its photoreaction is well characterized. In turn, a protein dimer is usually required to maintain its function as a PAS family domain. However, no partner protein for the signaling pathway of PYP has been identified yet. Here, we investigated the intermolecular interaction of PYP with a charged homopolypeptide, poly-L-lysine, in situ by combining chiral vibrational sum-frequency generation (VSFG) spectroscopy with molecular dynamics simulations and VSFG spectral calculations. Our combined experimental and theoretical approach enabled us to study the structure and orientation of proteins at interfaces based on probing the chiral N-H stretching and amide I and II vibrational modes. We found that PYP has a preferred orientation at the PLL interface, mainly due to dipole-dipole interaction, despite its water-soluble, i.e., cytoplasmic nature. Interestingly, the interaction surface and the orientation of PYP resulted in our model being nearly identical to a homodimer of PYP and a heterodimer in CNBh domains consisting of a PAS member. Our methodology provides a promising route for revealing orientational preferences of molecular groups during in situ protein-protein interactions and may ultimately help identify target proteins in PAS-domain signaling pathways.