Fluorescent protein-based probe architecture modulates rotational and translational diffusion readouts in polarization-dependent fluorescence correlation spectroscopy

We systematically engineered fluorescent protein-nanobody probes with different coupling rigidities and binding geometries to specific targets and analyzed the diffusion properties of their complexes by polarization-dependent fluorescence correlation spectroscopy (Pol-FCS). The results demonstrate that probe architecture critically affects Pol-FCS readouts, suggesting practical design principles for structure-sensitive probes. Polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) simultaneously measures translational and rotational diffusion of fluorescently labeled molecules and provides information on molecular size and shape. Rotational diffusion is, in principle, expected to provide even higher sensitivity than translational diffusion, which is routinely used in conventional FCS to detect binding of fluorescent protein (FP)-fused binders to their targets. However, when FPs are fused to binders via flexible linkers, local wobbling of the FP can decouple its rotational diffusion from that of the binding complex. Quantitative evaluation of this decoupling effect remains lacking. Here, leveraging our expertise from polarization-dependent orientation probes, POLArIS, we designed and compared two classes of probes, termed "Rigid" and "Flex," that differ in how tightly the FP is linked to the anti-ALFAtag nanobody, to evaluate how probe architecture affects Pol-FCS readouts. Using ALFAtag and the rod-like repeat protein DHR10 as a structurally anisotropic binding target, we varied probe binding geometry while keeping molecular weight nearly constant and measured rotational and translational diffusion. Flex probes exhibited shorter rotational diffusion times than Rigid probes when bound to the same targets, consistent with the expected local reorientation. Notably, translational diffusion times also showed architecture-dependent differences between Rigid and Flex probes. These results demonstrate that probe architecture is a key determinant of diffusion readouts in FP-based Pol-FCS and suggest practical probe design principles for sensitive reporters of intermolecular binding accompanied by changes in complex shape.