How Do Enzymes Orient When Trapped on Metal-Organic Framework (MOF) Surfaces?

Enzyme immobilization in metal-organic frameworks (MOFs) offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. Trapping enzymes on MOF surfaces would allow for catalysis involving large substrates. In both cases, the catalytic efficiency and selectivity depend not only on enzyme integrity/concentration but also orientation. However, it has been a challenge to determine the orientation of enzymes that are supported on solid matrices, which is even more challenging for enzymes immobilized/trapped in MOFs due to the interferences of the MOF background signals. To address such challenge, we demonstrate in this work the utilization of site-directed spin labeling in combination with Electron Paramagnetic Resonance spectroscopy, which allows for the first time the characterization of the orientation of enzymes trapped on MOF surfaces. The obtained insights are fundamentally important for MOF-based enzyme immobilization design and understanding enzyme orientation once trapped in solid matrices or even cellular confinement conditions.

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein-Polymer Hybrid.

Protein-polymer conjugates are attractive biomaterials which combine the functions of both proteins and polymers. The bioactivity of these hybrid materials, however, is often reduced upon conjugation. It is important to determine and monitor the protein structure and active site availability in order to optimize the polymer composition, attachment point, and abundance. The challenges in probing these insights are the large size and high complexity in the conjugates. Herein, we overcome the challenges by combining electron paramagnetic resonance (EPR) spectroscopy and atomic force microscopy (AFM) and characterize the structure of antibacterial hybrids formed by polyethylene glycol (PEG) and an antibacterial protein. We discovered that the primary reasons for activity loss were PEG blocking the substrate access pathway and/or altering protein surface charges. Our data indicated that the polymers tended to stay away from the protein surface and form a coiled conformation. The structural insights are meaningful for and applicable to the rational design of future hybrids.

Different Single-Enzyme Conformational Dynamics upon Binding Hydrolyzable or Nonhydrolyzable Ligands.

Single-molecule measurements of protein dynamics help unveil the complex conformational changes and transitions that occur during ligand binding and catalytic processes. Using high-resolution single-molecule nanocircuit techniques, we have investigated differences in the conformational dynamics and transitions of lysozyme interacting with three ligands: peptidoglycan substrate, substrate-based chitin analogue, and indole derivative inhibitors. While processing peptidoglycan, lysozyme followed one of the two mechanistic pathways for the hydrolysis of the glycosidic bonds: a concerted mechanism inducing direct conformational changes from open to fully closed conformations or a nonconcerted mechanism involving transient pauses in intermediate conformations between the open and closed conformations. In the presence of either chitin or an indole inhibitor, lysozyme was unable to access the fully closed conformation where catalysis occurs. Instead, lysozymes' conformational closures terminated at slightly closed, "excited" conformations that were approximately one-quarter of the full hinge-bending range. With the indole inhibitor, lysozyme reached this excited conformation in a single step without any evidence of rate-liming intermediates, but the same conformational motions with chitin involved three hidden, intermediate processes and features similar to the nonconcerted peptidoglycan mechanism. The similarities suggest that these hidden processes involve attempts to accommodate imperfectly aligned polysaccharides in the active site. The results provide a detailed glimpse of the enzyme-ligand interplay at the crux of molecular recognition, enzyme specificity, and catalysis.

Enzyme Immobilization on Graphite Oxide (GO) Surface via One-Pot Synthesis of GO/Metal-Organic Framework Composites for Large-Substrate Biocatalysis.

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often "harsh" reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal-Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.

A sulfonated mesoporous silica nanoparticle for enzyme protection against denaturants and controlled release under reducing conditions.

Mesoporous silica nanoparticles (MSiNPs) are attractive enzyme hosts, but current MSiNPs are limited by leaching, poor enzyme stabilization/protection, and difficulty in controlling enzyme release. Sulfonated MSiNPs are promising alternatives, but are challenged by narrow channels, lack of control over enzyme adsorption to particle surfaces, and controlled release of enzyme. By introducing amines on particle surfaces and sulfonate groups into the channels via disulfide bonds, we developed a unique sulfonated MSiNP to selectively encapsulate enzymes to the channels with enhanced enzyme stabilization under denaturing conditions. Via pore-expansion, the channel diameter was increased which allows for encapsulating nm-sized enzymes. This new concept/strategy to immobilize and deliver enzymes or other biomacromolecules were demonstrated using two model enzymes. Furthermore, we combine site-directed spin labeling with Electron Paramagnetic Resonance to confirm the enhanced enzyme-host interaction and reveal the preferred enzyme orientation in the channels. Lastly, the presence of disulfides allows for enzyme release under reducing conditions, a great potential for cancer treatments. To the best of our knowledge, this is the first report of sulfonated MSiNPs that simultaneously offer enhanced stability against denaturants and controlled release of enzymes under reducing conditions, with enzyme orientation resolved in the channels.

Real-time tracking of single-molecule collagenase on native collagen and partially structured collagen-mimic substrates.

The dynamic interactions of an individual matrix metalloproteinase-1 were imaged and monitored in the presence of either triple-helical or non-triple-helical, partially structured collagen-mimic substrates. The enzyme exhibited ten-fold increased catalytic turnover rates with the structurally modified substrate by skipping the triple-helix unwinding step during the catalytic pathway.

Probing the structural basis and adsorption mechanism of an enzyme on nano-sized protein carriers.

Silica nanoparticles (SiNPs) are important nano-sized, solid-state carriers/hosts to load, store, and deliver biological or pharmaceutical cargoes. They are also good potential solid supports to immobilize proteins for fundamental protein structure and dynamics studies. However, precaution is necessary when using SiNPs in these areas because adsorption might alter the activity of the cargoes, especially when enzymes are loaded. Therefore, it becomes important to understand the structural basis of the cargo enzyme activity changes, if there is any. The high complexity and dynamics of the nano-bio interface present many challenges. Reported here is a comprehensive study of the structure, dynamics, and activity of a model enzyme, T4 lysozyme, upon adsorption to a few surface-modified SiNPs using several experimental techniques. Not surprisingly, a significant activity loss on each studied SiNP was found. The structural basis of the activity loss was identified based on results from a unique technique, the Electron Paramagnetic Resonance (EPR) spectroscopy, which probes structural information regardless of the complexity. Several docking models of the enzyme on SiNPs with different surfaces, at different enzyme-to-SiNP ratios are proposed. Interestingly, we found that the adsorbed enzyme can be desorbed via pH adjustment, which highlighted the potential to use SiNPs for enzyme/protein delivery or storage due to the high capacity. In order to use SiNPs as enzyme hosts, minimizing the enzymatic activity loss upon adsorption is needed. Lastly, the work outlined here demonstrate the use of EPR in probing structural information on the complex (inorganic)nano-bio interface.