Correlating Structural and Functional Heterogeneity of Immobilized Enzymes.

Many nanobiotechnology applications rely on stable and efficient integration of functional biomacromolecules with synthetic nanomaterials. Unfortunately, the reasons for the ubiquitous loss of activity of immobilized enzymes remain poorly understood due to the difficulty in distinguishing between distinct molecular-level mechanisms. Here, we employ complementary single-molecule fluorescence methods that independently measure the impact of immobilization on the structure and function ( i. e., substrate binding kinetics) of nitroreductase (NfsB). Stochastic statistical modeling methods were used to unambiguously quantify the effects of immobilized NfsB structural dynamics on function, allowing us to explicitly separate effects due to conformation and accessibility. Interestingly, we found that nonspecifically tethered NfsB exhibited enhanced stability compared to site-specifically tethered NfsB; however, the folded state of site-specifically tethered NfsB had faster substrate binding rates, suggesting improved active site accessibility. This demonstrated an unexpected intrinsic trade-off associated with competing bioconjugation methods, suggesting that it may be necessary to balance conformational stability versus active site accessibility. This nuanced view of the impact of immobilization will facilitate a rational approach to the integration of enzymes with synthetic nanomaterials.