Dynamic conformations of fluorophores on self-labeling protein tags.

Self-labeling protein tags (SLPs) enable protein-specific conjugation of synthetic fluorophores for versatile bioimaging, yet their rational engineering has largely relied on static structural models. Here, we reveal that crystal structures of SLP-dye complexes generally bear packing artifacts due to strong π-π interactions of dyes, frequently failing to profile the chromophore microenvironment. By integrating molecular dynamics (MD) with crystallography, we elucidate distinct dynamic behaviors of fluorophores across major SLPs: quasi-free diffusion in SNAP-tag, balanced hydrophobic-polar interactions in TMP-tag, and discrete, state-selective anchoring in HaloTag and Rho-tag. Comparisons with the newly developed SNAP-tag2 demonstrate that functional evolution reshapes conformational landscapes rather than merely strengthening binding. We further validate simulation-derived predictions through MD-guided lattice selection. Together, this work establishes a comparative, dynamic framework for understanding SLP-dye interactions beyond crystallography, while providing a conceptual basis for further engineering chemigenetic tools for advanced imaging and sensing.