Electromechanical Photophysics of GFP Packed Inside Viral Protein Cages Probed by Force-Fluorescence Hybrid Single-Molecule Microscopy.

Packing biomolecules inside virus capsids has opened new avenues for the study of molecular function in confined environments. These systems not only mimic the highly crowded conditions in nature, but also allow their manipulation at the nanoscale for technological applications. Here, green fluorescent proteins are packed in virus-like particles derived from P22 bacteriophage procapsids. The authors explore individual virus cages to monitor their emission signal with total internal reflection fluorescence microscopy while simultaneously changing the microenvironment with the stylus of atomic force microscopy. The mechanical and electronic quenching can be decoupled by ≈10% each using insulator and conductive tips, respectively. While with conductive tips the fluorescence quenches and recovers regardless of the structural integrity of the capsid, with the insulator tips quenching only occurs if the green fluorescent proteins remain organized inside the capsid. The electronic quenching is associated with the coupling of the protein fluorescence emission with the tip surface plasmon resonance. In turn, the mechanical quenching is a consequence of the unfolding of the aggregated proteins during the mechanical disruption of the capsid.