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1. On the interface of enzyme and spatial confinement: The impacts of confinement rigidity, shape, and surface properties on the interplay of enzyme structure, dynamics, and function

   https://doi.org/10.1063/5.0167117  

   Li, Qiaobin; Armstrong, Zoe; MacRae, Austin; Lenertz, Mary; Feng, Li; Yang, Zhongyu (December 2023, Chemical Physics Reviews)

   Confining proteins in synthetic nanoscale spatial compartments has offered a cell-free avenue to understand enzyme structure–function relationships and complex cellular processes near the physiological conditions, an important branch of fundamental protein biophysics studies. Enzyme confinement has also provided advancement in biocatalysis by offering enhanced enzyme reusability, cost-efficiency, and substrate selectivity in certain cases for research and industrial applications. However, the primary research efforts in this area have been focused on the development of novel confinement materials and investigating protein adsorption/interaction with various surfaces, leaving a fundamental knowledge gap, namely, the lack of understanding of the confined enzymes (note that enzyme adsorption to or interactions with surfaces differs from enzyme confinement as the latter offers an enhanced extent of restriction to enzyme movement and/or conformational flexibility). In particular, there is limited understanding of enzymes' structure, dynamics, translocation (into biological pores), folding, and aggregation in extreme cases upon confinement, and how confinement properties such as the size, shape, and rigidity affect these details. The first barrier to bridge this gap is the difficulty in “penetrating” the “shielding” of the confinement walls experimentally; confinement could also lead to high heterogeneity and dynamics in the entrapped enzymes, challenging most protein-probing experimental techniques. The complexity is raised by the variety in the possible confinement environments that enzymes may encounter in nature or on lab benches, which can be categorized to rigid confinement with regular shapes, rigid restriction without regular shapes, and flexible/dynamic confinement which also introduces crowding effects. Thus, to bridge such a knowledge gap, it is critical to combine advanced materials and cutting-edge techniques to re-create the various confinement conditions and understand enzymes therein. We have spearheaded in this challenging area by creating various confinement conditions to restrict enzymes while exploring experimental techniques to understand enzyme behaviors upon confinement at the molecular/residue level. This review is to summarize our key findings on the molecular level details of enzymes confined in (i) rigid compartments with regular shapes based on pre-formed, mesoporous nanoparticles and Metal–Organic Frameworks/Covalent-Organic Frameworks (MOFs/COFs), (ii) rigid confinement with irregular crystal defects with shapes close to the outline of the confined enzymes via co-crystallization of enzymes with certain metal ions and ligands in the aqueous phase (biomineralization), and (iii) flexible, dynamic confinement created by protein-friendly polymeric materials and assemblies. Under each case, we will focus our discussion on (a) the way to load enzymes into the confined spaces, (b) the structural basis of the function and behavior of enzymes within each compartment environments, and (c) technical advances of our methodology to probe the needed structural information. The purposes are to depict the chemical physics details of enzymes at the challenging interface of natural molecules and synthetic compartment materials, guide the selection of enzyme confinement platforms for various applications, and generate excitement in the community on combining cutting-edge technologies and synthetic materials to better understand enzyme performance in biophysics, biocatalysis, and biomedical applications. 

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2. Agarose‐based 3D Cell Confinement Assay to Study Nuclear Mechanobiology

   https://doi.org/10.1002/cpz1.847  

   Elpers, Margaret A; Varlet, Alice‐Anais; Agrawal, Richa; Lammerding, Jan (July 2023, Current Protocols)

   Abstract Cells in living tissues are exposed to substantial mechanical forces and constraints imposed by neighboring cells, the extracellular matrix, and external factors. Mechanical forces and physical confinement can drive various cellular responses, including changes in gene expression, cell growth, differentiation, and migration, all of which have important implications in physiological and pathological processes, such as immune cell migration or cancer metastasis. Previous studies have shown that nuclear deformation induced by 3D confinement promotes cell contractility but can also cause DNA damage and changes in chromatin organization, thereby motivating further studies in nuclear mechanobiology. In this protocol, we present a custom‐developed, easy‐to‐use, robust, and low‐cost approach to induce precisely defined physical confinement on cells using agarose pads with micropillars and externally applied weights. We validated the device by confirming nuclear deformation, changes in nuclear area, and cell viability after confinement. The device is suitable for short‐ and long‐term confinement studies and compatible with imaging of both live and fixed samples, thus presenting a versatile approach to studying the impact of 3D cell confinement and nuclear deformation on cellular function. This article contains detailed protocols for the fabrication and use of the confinement device, including live cell imaging and labeling of fixed cells for subsequent analysis. These protocols can be amended for specific applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Design and fabrication of the confinement device wafer Basic Protocol 2: Cell confinement assay Support Protocol 1: Fixation and staining of cells after confinement Support protocol 2: Live/dead staining of cells during confinement 

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3. Engineering a Mesoporous Silicon Nanoparticle Cage to Enhance Performance of a Phosphotriesterase Enzyme for Degradation of VX Nerve Agent

   https://doi.org/10.1002/advs.202409535  

   Lu, Yi‐Sheng; Moreno, Eduardo_Reynoso; Huang, Yubin; Fan, Ruhan; Tucker, Ashley_T; Wright, Linnzi_K; Evans, Ronald_A; Ahern, Brooke_M; Owens, Donald_E; Chappell, Stephen_A; et al (November 2024, Advanced Science)

   Abstract The organophosphate (OP)‐hydrolyzing enzyme phosphotriesterase (PTE, variant L7ep‐3a) immobilized within a partially oxidized mesoporous silicon nanoparticle cage is synthesized and the catalytic performance of the enzyme@nanoparticle construct for hydrolysis of a simulant, dimethyl p‐nitrophenyl phosphate (DMNP), and the live nerve agent VX is benchmarked against the free enzyme. In a neutral aqueous buffer, the optimized construct shows a ≈2‐fold increase in the rate of DMNP turnover relative to the free enzyme. Enzyme@nanoparticles with more hydrophobic surface chemistry in the interior of the pores show lower catalytic activity, suggesting the importance of hydration of the pore interior on performance. The enzyme@nanoparticle construct is readily separated from the neutralized agent; the nanoparticle is found to retain DMNP hydrolysis activity through seven decontamination/recovery cycles. The nanoparticle cage stabilizes the enzyme against thermal denaturing and enzymatic (trypsin) degradation conditions relative to free enzyme. When incorporated into a topical gel formulation, the PTE‐loaded nanoparticles show high activity toward the nerve agent VX in an ex vivo rabbit skin model. In vitro acetylcholinesterase (AChE) assays in human blood show that the enzyme@nanoparticle construct decontaminates VX, preserving the biological function of AChE when exposed to an otherwise incapacitating dose. 

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4. The gas|liquid interface eclipses the liquid|liquid interface for glucose oxidase rate acceleration in microdroplets

   https://doi.org/10.1073/pnas.2416353121  

   Krushinski, Lynn E; Herchenbach, Patrick J; Dick, Jeffrey E (December 2024, Proceedings of the National Academy of Sciences)

   The curious chemistry observed in microdroplets has captivated chemists in recent years and has led to an investigation into their ability to drive seemingly impossible chemistries. One particularly interesting capability of these microdroplets is their ability to accelerate reactions by several orders of magnitude. While there have been many investigations into which reactions can be accelerated by confinement within microdroplets, no study has directly compared reaction acceleration at the liquid|liquid and gas|liquid interfaces. Here, we confine glucose oxidase, one of life’s most important enzymes, to microdroplets and monitor the turnover rate of glucose by the electroactive cofactor, hexacyanoferrate (III). We use stochastic electrochemistry to monitor the collision of single femtoliter water droplets on an ultramicroelectrode. We also develop a measurement modality to robustly quantify reaction rates for femtoliter liquid aerosol droplets, where the majority of the interface is gas|liquid. We demonstrate that the gas|liquid interface accelerates enzyme turnover by over an order of magnitude over the liquid|liquid interface. This is the first apples-to-apples comparison of reaction acceleration at two distinct interfaces that indicates that the gas|liquid interface plays a central role in driving curious chemistry. 

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5. The effect of axisymmetric confinement on propulsion of a three-sphere microswimmer

   https://doi.org/10.1063/5.0163348  

   Gürbüz, Ali; Lemus, Andrew; Demir, Ebru; Pak, On_Shun; Daddi-Moussa-Ider, Abdallah (August 2023, Physics of Fluids)

   Swimming at the microscale has recently garnered substantial attention due to the fundamental biological significance of swimming microorganisms and the wide range of biomedical applications for artificial microswimmers. These microswimmers invariably find themselves surrounded by different confining boundaries, which can impact their locomotion in significant and diverse ways. In this work, we employ a widely used three-sphere swimmer model to investigate the effect of confinement on swimming at low Reynolds numbers. We conduct theoretical analysis via the point-particle approximation and numerical simulations based on the finite element method to examine the motion of the swimmer along the centerline in a capillary tube. The axisymmetric configuration reduces the motion to one-dimensional movement, which allows us to quantify how the degree of confinement affects the propulsion speed in a simple manner. Our results show that the confinement does not significantly affect the propulsion speed until the ratio of the radius of the tube to the radius of the sphere is in the range of O(1)−O(10), where the swimmer undergoes substantial reduction in its propulsion speed as the radius of the tube decreases. We provide some physical insights into how reduced hydrodynamic interactions between moving spheres under confinement may hinder the propulsion of the three-sphere swimmer. We also remark that the reduced propulsion performance stands in stark contrast to the enhanced helical propulsion observed in a capillary tube, highlighting how the manifestation of confinement effects can vary qualitatively depending on the propulsion mechanisms employed by the swimmers. 

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