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Using the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) main protease (M\n                    <jats:sup>pro<\/jats:sup>\n                    ) as a biologically and clinically relevant model, we demonstrate that subtle changes in the phenylalanine benzene ring breathing (BRB) mode serve as a precise spectroscopic indicator of \u03c0-\u03c0 stacking strength. This signal is highly responsive to both protein concentration-dependent dimerization and ligand-induced structural changes. M\n                    <jats:sup>pro<\/jats:sup>\n                    forms a catalytically active dimer stabilized by a conserved aromatic triad (phenylalanine-140, histidine-163, and histidine-172), providing an ideal system to interrogate \u03c0-stacking at an important protein interface. Potent inhibitors MPI8 and nirmatrelvir produced the strongest BRB spectral shifts, broadening, and intensity changes, consistent with enhanced aromatic stacking and dimer stabilization, whereas halicin and VB-B-145 showed weaker engagement. BRB spectral changes also showed quantitative correlation with dimerization efficiency, published IC\n                    <jats:sub>50<\/jats:sub>\n                    (median inhibitory concentration) values, and antiviral efficacy in A549-ACE2 cells. Complementary density functional theory revealed electron density rearrangements and vibrational coupling patterns unique to stacked aromatic residues. This integrated spectroscopic-computational approach enables quantitative probing of \u03c0-\u03c0 stacking in native-like protein environments and positioning TRIP as a generalizable tool for designing drugs targeting aromatic protein-protein interfaces.\n                  <\/jats:p>","DOI":"10.1126\/sciadv.aeb3917","type":"journal-article","created":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T18:00:23Z","timestamp":1775671223000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":2,"title":["From vibrations to function: Spectroscopic detection and quantification of \u03c0-\u03c0 stacking in drug-responsive protein complexes"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1617-3427","authenticated-orcid":true,"given":"Narangerel","family":"Altangerel","sequence":"first","affiliation":[{"name":"Texas A&amp;M University, College Station, TX 77843, 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