Multivalent Interactions between Chaperone and Ribosome-Nascent Chain Complex Revealed by High-Speed AFM and MD Simulations

Trigger Factor (TF) is a primary ATP-independent molecular chaperone in bacteria that engages nascent polypeptide chains emerging from the ribosomal exit tunnel to assist their folding. However, the real-time behavior of TF during active translation under near-physiological conditions remains elusive. Here, we employ high-speed atomic force microscopy (HS-AFM) imaging to visualize TF dynamics on intact Escherichia coli ribosomes in real time. We observe that TF transitions between compact and extended conformations and forms stable and transient contacts near ribosomal proteins uL23 and bL17, respectively. Interestingly, TFs engage distinct regions of the same ribosome-nascent chain complex, with one TF binding near the nascent chain and another near bL17, revealing multivalent interactions on the ribosome surface. Complementary all-atom molecular dynamics simulations reproduced the observed TF conformations and interaction dynamics, validating the experimentally observed structural transitions and dual-site engagement. This integrative approach uncovers previously inaccessible dynamics of ribosome-associated chaperones and offers a broadly applicable platform to probe cotranslational folding under near-physiological conditions.