Fig. 1.

Workflow for determining CP-substrate interactions via Raman spectroscopy. An alkyne-labeled fatty acid (of selected length) is ligated to the terminal thiol of the Ppant arm via the promiscuous ligase AasS (top). The probe-labeled molecular cargo serves as a reporter of whether a substrate is sequestered into the hydrophobic cavity of the CP through changes in the Raman scattering spectrum. The C≡C frequency reports on the solvation environment (lower frequency when the probe is in an aqueous environment, or higher frequency when the probe is in the protein’s hydrophobic cavity) and the line shape reports on the ps-resolved range of conformations