Investigating the Impact of Polymer Functional Groups on the Stability and Activity of Lysozyme-Polymer Conjugates.

Polymers are often conjugated to proteins to improve stability; however, the impact of polymer chain length and functional groups on protein structure and function is not well understood. Here we use RAFT polymerization to grow polymers of different lengths and functionality from a short acrylamide oligomer with a RAFT end group conjugated to lysozyme. We show by X-ray crystallography that enzyme structure is minimally impacted by modification with the RAFT end group. Significant activity toward the negatively charged Micrococcus lysodeicticus cell wall was maintained when lysozyme was modified with cationic polymers. Thermal and chemical stability of the conjugates was characterized using differential scanning fluorimetry and tryptophan fluorescence. All conjugates had a lower melting temperature; however, conjugates containing ionic or substrate mimicking polymers were more resistant to denaturation by guanidine hydrochloride. Our results demonstrate that tailoring polymer functionality can improve conjugate activity and minimize enzymatic inactivation by denaturants.

Multisite clickable modification of proteins using lipoic acid ligase.

Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

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.

Stabilization of Immobilized Enzymes via the Chaperone-Like Activity of Mixed Lipid Bilayers.

Biomimetic lipid bilayers represent intriguing materials for enzyme immobilization, which is critical for many biotechnological applications. Here, through the creation of mixed lipid bilayers, the retention of immobilized enzyme structures and catalytic activity are dramatically enhanced. The enhancement in the retention of enzyme structures, which correlated with an increase in enzyme activity, is observed using dynamic single-molecule (SM) fluorescence methods. The results of SM analysis specifically show that lipid bilayers composed of mixtures of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl- sn-glycero-3-phospho-(1'- rac-glycerol) (DOPG) stabilize the folded state of nitroreductase (NfsB), increasing the rate of refolding relative to unfolding of enzyme molecules on the bilayer surface. Remarkably, for optimal compositions with 15-50% DOPG, over 95% of NfsB remains folded while the activity of the enzyme is increased as much as 2 times over that in solution. Within this range of DOPG, the strength of the interaction of folded and unfolded NfsB with the bilayer surface was also significantly altered, which was evident by the change in the diffusion of folded and unfolded NfsB in the bilayer. Ultimately, these findings provide direct evidence for the chaperone-like activity of mixed DOPG/DOPC lipid bilayers, which can be controlled by tuning the fraction of DOPG in the bilayer.

Approaches for Conjugating Tailor-Made Polymers to Proteins.

A series of methods are outlined for attaching functional polymers to proteins. Polymers with good control over structure, functionality, and composition can be created using reversible addition-fragmentation chain transfer (RAFT) polymerization. These polymers can be covalently linked to enzymes and proteins using either the "grafting-to" approach, where a preformed polymer is attached to the protein surface, or the "grafting-from" approach, where the polymer is grown from the protein surface. Methods for grafting-to, or attaching the RAFT chain transfer agent to the protein surface outlined include the commonly used carbodiimide/activated ester (EDC/NHS) coupling. Methods are also outlined to graft-from the surface of the protein using RAFT polymerization. Additionally, it is possible to site specifically introduce a reactive azide group to the protein surface using enzymatic ligation as a posttranslational modification. This reactive azide group can be conjugated to an alkyne-containing polymer using highly efficient click chemistry. These robust protocols can produce protein-polymer conjugates with various architectures and functionalities. Methods are also outlined for characterization of the resulting bioconjugates.

The best of both worlds: active enzymes by grafting-to followed by grafting-from a protein.

Hydrophilic polymers were attached to lysozyme by a combination of grafting-to and grafting-from approaches using RAFT polymerization. A hydrophilic oligomer was synthesized, and attached to the protein. The protein-oligomer hybrid contained the RAFT end group, enabling chain extension in solution. Lysozyme maintained activity throughout this process.