Elucidating the Dynamics of Nanoparticle‐protein Interaction at a Biomolecular Level: Structural and Functional Studies Using Firefly Luciferase

A disparity exists between the physicochemical properties exhibited by nanoparticles (NPs) and how this relates to physiological manifestations when applied to biological systems. An abundance of research exists on how NP‐protein interactions, especially biologically relevant NPs, can directly mediate physiological processes via alterations in protein‐protein, protein‐nucleic acid, and enzyme activity, but directly relating the physicochemical and structural changes to functional activity is less defined. In the current study we investigated the effect of biologically relevant NPs (magnesium (II) oxide (MgO), zinc (II) oxide (ZnO), copper (Cu), nickel oxide (NiO), iron (III) oxide (Fe 2 O 3 ), and boron carbide (B 4 C)) on a model enzyme, firefly luciferase. B 4 C was incorporated because of previous studies revealing its drastic inhibitory effect on luciferase. Luciferase has inherent bioluminescence properties, catalyzing the production of light by the oxidation of a D‐luciferin substrate. The dependence of protein functionality requires the maintenance of the active conformation by the hydrophobic, covalent, and electrostatic interactions between chemical moieties. In the case of enzymes, minor disturbances of the enzyme (Luciferase), substrate (D‐luciferin), or enzyme‐substrate complexes can lead to complete loss of catalytic activity, however, the underlying nature of the relationship between the structure and function remains heavily disputed. The intrinsic fluorescence of luciferase, ascribed to Tryptophan residues, revealed a concentration‐dependent quenching for most, but not all NPs. Of the NPs tested, B 4 C and Cu NPs revealed the most significant changes, both causing significant quenching at higher concentration (≥ 75% at 50 μg/ml). More importantly, when comparing the fluorescence maximum relative to overall fluorescence, as an indicator of the microstate populations that exists within luciferase, Cu and B 4 C NPs revealed a clear deviation from the normal fluorescence indicative of a conformational change in luciferase and NP‐complexation. Bioluminescence analysis revealed kinetic changes by Cu, B 4 C, and Fe 2 O 3 NPs, with complete inhibition by B 4 C and destabilization of the enzyme‐substrate complex by Cu and Fe 2 O 3 . Limited proteolysis experiments on luciferase with and without NP complexes showed that NiO and B 4 C decreased susceptibility to proteolysis, but Cu, MgO, and ZnO increased proteolytic sensitivity (Cu > MgO > ZnO) suggesting a more globular (NiO, B 4 C) and denatured conformational state (Cu, MgO, ZnO). Finally, because the bioluminescence phenomena is a result of an enzyme‐substrate reaction, D‐luciferin fluorescence was analyzed in the presence of NPs, which indicated substrate level interaction by ZnO and Cu. Collectively, these data suggest the role of protein‐NP interaction and the structural and functional alterations of luciferase, with distinctive alterations by Cu, Fe 2 O 3 , MgO and B 4 C NPs, providing a link between structural perturbation and functionality. Support or Funding Information This investigation was supported by the Undergraduate Summer Research Fellowship (American Physiological Society) and by a research grant from the National Cancer Institute ( 7R15CA139390‐03 ) to RKD.