Molecular crowding differentially regulates magnetic field-dependent fluorescence, recovery kinetics, and photostability of the magnetosensitive protein MagLOV2

Macromolecular crowding and solvent composition can alter protein conformation, diffusion, hydration, and cofactor photophysics, yet their influence on magnetically sensitive flavoproteins is poorly understood. We tested seven molecular environments in MagLOV2-overexpressing Escherichia coli lysate using a custom-built, high-intensity magnetofluorescence platform. Five-point concentration series of Dextran 70, Ficoll PM70, PEG6000, PEG3350, glycerol, ethylene glycol, and bovine serum albumin (BSA) were measured at 22 mT under continuous 6.12 W/cm2 blue-light excitation. Dextran 70, Ficoll PM70, and PEG6000 progressively attenuated the negative magnetic field effect (MFE), whereas PEG3350 shifted toward mild enhancement at higher concentrations. Glycerol and ethylene glycol enhanced the negative MFE despite producing the strongest slowing of recovery upon the field being switched off (OFF-recovery), while BSA modestly enhanced MFE and accelerated OFF-recovery. Across 105 treatment cuvettes, the change in MFE was not globally correlated with changes in the OFF-recovery time constant (r = −0.07, p = 0.449). The initial pre-field illumination period revealed a second phenotype: Dextran and Ficoll increased late fluorescence loss, whereas glycerol and BSA were photoprotective. Pre-field fluorescence loss correlated moderately with MFE change across the full dataset (r = 0.474, p = 3.28 × 10 −7 ) but not consistently within crowder classes. These findings show that MagLOV2 spin-dependent magnetofluorescence is shaped by crowder chemistry, polymer size, solvent environment, recovery kinetics, and photostability rather than by a single generic crowding or viscosity mechanism.