Many nanobiotechnology applications rely on stable and efficient integration of functional biomacromolecules with synthetic nanomaterials. Unfortunately, the reasons for the ubiquitous loss of function of immobilized enzymes remain poorly understood due to the difficulty in distinguishing between distinct molecular-level mechanisms. Here, we employ complementary single molecule (SM) methods that independently measure the structure and function (i.e., substrate binding kinetics) of nitroreductase (NfsB) that was immobilized using different bioconjugation strategies. The folding state was monitored using SM Förster resonance energy transfer, while fluorescence co-localization microscopy was used to measure SM substrate binding rates, providing a detailed picture of heterogeneous conformational dynamics and activity. Stochastic statistical modeling methods were used to unambiguously quantify the effects of enzyme structural dynamics on enzyme function, allowing us to explicitly separate effects due to conformation and accessibility. Interestingly, we found that non-specifically tethered enzymes exhibited enhanced structural stability compared to site-specifically tethered enzymes; however, the folded state of site-specifically tethered enzymes was more active. 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 enzyme immobilization will facilitate a rational approach to the integration of enzymes with synthetic nanomaterials.

Correlating structural and functional heterogeneity of immobilized enzymes

256th National Meeting (2018)

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