(570f) Post-Translational Azide Functionalization for Optimizing Immobilized Protein Properties

Site-specific azide functionalization of proteins allows for diverse chemical modification via azide-alkyne click chemistry and thus holds high potential for pharmaceutical and industrial protein applications. The introduction of azide groups benefits from post-translational techniques, which allow for precise temporal control of azide functionalization, preventing undesirable azide reduction reactions and, ultimately, improving the homogeneity of modified protein populations. One promising approach to post-translational azide functionalization entails the use of the enzyme, lipoic acid ligase, which covalently and site-specifically attaches an azide group to a short peptide recognition sequence known as the LAP sequence. We have found that this sequence, though traditionally used as an N- or C-terminal protein tag, may be inserted at numerous internal sites within the model protein, GFP, as well as within the industrially relevant enzyme, lipase A, without negatively affecting structure or function or significantly reducing expression levels. Azide attachment to these internal recognition sequences proceeds efficiently, with a high degree of temporal control and population homogeneity. Given the flexibility and robust nature of this approach, we have utilized internal LAP insertions within lipase for orientation-controlled immobilization to alkyne-functionalized silica nanoparticles. Specifically, we are observing the structural impact of different immobilized protein orientations on these nanoparticles through circular dichroism and coupling these assays with activity and stability measurements. Combined, these experiments allow us to identify azide attachment sites that yield optimal protein properties upon immobilization. Ultimately, exploring this approach to optimize protein function at material interfaces may yield a general strategy for designing enzyme-functionalized materials.