Fluorescent protein-based probe architecture modulates rotational and translational diffusion readouts in...

Area of Science:

• Biophysics
• Molecular Biology
• Spectroscopy

Background:

• Polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) measures molecular diffusion and provides insights into size and shape.
• Fluorescent protein (FP)-fused binders are used to detect molecular binding, but flexible linkers can decouple FP rotation from complex rotation.
• Quantitative evaluation of this decoupling effect in FP-based probes is lacking.

Purpose of the Study:

• To systematically engineer fluorescent protein-nanobody probes with varying coupling rigidities and binding geometries.
• To analyze the diffusion properties of probe-target complexes using Pol-FCS.
• To establish practical design principles for structure-sensitive probes.

Main Methods:

• Designed and compared two classes of probes: 'Rigid' and 'Flex', differing in FP-nanobody linkage.
• Utilized anti-ALFAtag nanobodies and the rod-like protein DHR10 as a binding target.
• Employed polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) to measure translational and rotational diffusion.

Main Results:

• Probe architecture critically affects Pol-FCS readouts.
• Flex probes exhibited shorter rotational diffusion times than Rigid probes, indicating local FP reorientation.
• Translational diffusion times also showed architecture-dependent differences between Rigid and Flex probes.

Conclusions:

• Probe architecture is a key determinant of diffusion readouts in FP-based Pol-FCS.
• Design principles for FP-nanobody probes can enhance sensitivity for detecting binding events.
• These findings enable the development of sensitive reporters for intermolecular binding and shape changes.