Nanomechanical interactions of phenylalanine–glycine nucleoporins studied by single molecule force–volume spectroscopy

Phenylalanine–glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-β. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-β, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-β-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG–receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG–receptor interactions instead of FG–FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force–volume (FV) mode.