Engineering the Composition of Heterogeneous Lipid Bilayers to Stabilize Tethered Enzymes

Abstract

As surfaces for enzyme immobilization, lipid bilayers (LBs) have the potential to exhibit chaperone‐like activity, which can greatly enhance enzyme stability. Here, it is shown that this stabilizing effect can be generalized to a broad range of enzymes by modifying the mixed lipid composition based on the properties of the enzyme. This is demonstrated by analyzing the stability of nitroreductase, lipase, organophosphorus hydrolase, and lysozyme on LBs composed by mixing zwitterionic 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine with anionic 1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) or cationic 1,2‐dioleoyl‐sn‐glycero‐3‐ethylphosphocholine. Interestingly, while the optimal lipid composition exhibits a zeta potential with the same sign as each enzyme, the stabilizing compositional ranges are broad, and mixed lipid LBs are more stabilizing than homogeneous LBs with similar charge. This suggests that net charge and compositional heterogeneity are both important factors. Moreover, the degree of stabilization on appropriate bilayers under extremely denaturing conditions (7 m urea) is unprecedented, and activity of the enzymes is up to three‐fold greater than that of the soluble enzyme at each enzyme's temperature optimum. By correlating enzyme diffusion on LBs with information about re‐folding kinetics derived from single‐molecule measurements, the influence of charge and composition on enzyme‐LB interactions is shown, providing a rationale for the stabilizing effect of heterogeneous LBs.

Cadherin clusters stabilized by a combination of specific and nonspecific cis-interactions

“…This was done by constructing complementary cumulative squared displacement distributions (CCSDDs) for each state, under each experimental condition, and then fitting these distributions to a Gaussian mixture model containing three terms (See Materials and methods section for more details on distribution calculations, fitting, and calculation). represents the average instantaneous molecular diffusion coefficient at the shortest experimentally accessible time scale and is especially useful for systems where molecules change FRET states within a trajectory ( Chaparro Sosa et al, 2020 ; Chaparro Sosa et al, 2018 ; Langdon et al, 2015 ). Additionally, overall CCSDDs were constructed, in order to determine overall values of under each experimental condition.…”

Mixed Phospholipid Vesicles Catalytically Inhibit and Reverse Amyloid Fibril Formation

“…We previously demonstrated that the stability and activity of enzymes tethered to mixed lipid bilayers was directly related to lipid composition. Furthermore, using single-molecule methods, we found that that the enhancement in enzyme stability and activity upon varying lipid composition was due to a chaperone-like effect of the bilayer, whereby the bilayer actively mediated the refolding of denatured enzyme molecules at the bilayer–solution interface. , We therefore hypothesize that tuning the composition of mixed DOPG/DOPC vesicles may have a similar effect on the stabilization of Aβ. Specifically, by acting as a molecular chaperone, the mixed DOPG/DOPC vesicles may degrade Aβ fibrils and, moreover, catalyze Aβ refolding.…”

“…Here, we studied the interactions between Aβ peptides/fibrils and unilamellar vesicles composed of mixtures of zwitterionic and anionic phospholipids, specifically 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl- sn -glycero-3-phosphoglycerol (DOPG), respectively. This choice was motivated by our previous observations that mixed lipid bilayers had remarkable stabilizing effects on enzymes tethered to lipid bilayers, especially when the bilayer and protein exhibited like net electrical charges . While studying the interaction of mixed DOPG/DOPC vesicles with Aβ peptides/fibrils, we investigated the effect of vesicle composition on both the prevention of fibrillation in solution, as well as the disruption of preformed fibrillar aggregates.…”

Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity

“…Recent findings from dynamic single-molecule (SM) experiments have significantly altered our view of how the functional utility of enzymes upon immobilization may be improved. − Based on these findings, it has become apparent that both long-term stability and the immediate retention of enzyme activity and structure during immobilization to surfaces are critical. Importantly, because long-term stability and immediate retention of activity and structure involve different mechanisms, different strategies may be required to address each of these aspects.…”

“…For example, while the long-term stability of immobilized enzymes may be enhanced by tethering enzymes to materials that stabilize the native state of the enzyme and/or promote refolding of denatured enzyme molecules, the immediate retention of activity and structure may be enhanced by controlling the exploration of the surface by the enzyme prior to reaction with the material. Although there has been extensive work on improving the long-term stability of immobilized enzymes, ,− preventing the immediate denaturation of enzymes has received far less attention.…”