Monitoring Enzyme Clustering in a Highly Crystalline Metal–Organic Framework by Small-Angle Neutron Scattering

Xiaoliang Wang; Lilin He; Shuo Qian; Shengyi Su; Omar K. Farha; Shengqian Ma

doi:10.1002/anie.202522967

Monitoring Enzyme Clustering in a Highly Crystalline Metal–Organic Framework by Small-Angle Neutron Scattering

Xiaoliang Wang
, Lilin He
, Shuo Qian
, Shengyi Su
, Omar K. Farha
, Shengqian Ma

Research output: Contribution to journal › Article › peer-review

Abstract

The molecular-level investigation of enzyme behavior in confined, cell-free environments is essential to understanding intrinsic properties and optimizing systems for desired functions. Metal–organic frameworks (MOFs) provide unique structural features that enable the immobilization of bulky biomolecules and allow direct probing of enzymatic behavior under confinement. Here, small-angle neutron scattering (SANS) was employed to probe porosity changes in a highly crystalline Tb-mesoMOF and to reveal the spatial arrangement of encapsulated enzymes across long-range length scales. Structural characteristics such as framework void space were resolved by SANS, while contrast-matching experiments using D₂O/H₂O mixtures suppressed background scattering from the MOF and isolated the enzyme contribution. Compared to unloaded Tb-mesoMOF, cytochrome c (Cyt. c)-loaded Tb-mesoMOF exhibited the emergence of a broad scattering feature at low q (∼0.005 Å⁻¹), indicative of enzyme clustering within the framework, accompanied by enhanced loading rate and capacity. Additional structural analyses using complementary techniques further corroborated these findings.

Original language	English
Article number	e22967
Journal	Angewandte Chemie - International Edition
Volume	65
Issue number	4
DOIs	https://doi.org/10.1002/anie.202522967
State	Published - Jan 22 2026

Funding

The authors acknowledge the financial support from the US National Science Foundation (DMR-1352065) and the Robert A. Welch Foundation (B-0027). Partial support from U.S. Army Contracting Command (W52P1J-21–9–3023) is also acknowledged (OKF). Neutron scattering experiments on Bio-SANS were supported by the Center for Structural Molecular Biology funded by DOE Biological and Environmental Research (ERKP291). A portion of this research used resources at the High Flux Isotope Reactor, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory (IPTS-27697, IPTS-18982 and IPTS-20263). The authors acknowledge the financial support from the US National Science Foundation (DMR‐1352065) and the Robert A. Welch Foundation (B‐0027). Partial support from U.S. Army Contracting Command (W52P1J‐21–9–3023) is also acknowledged (OKF). Neutron scattering experiments on Bio‐SANS were supported by the Center for Structural Molecular Biology funded by DOE Biological and Environmental Research (ERKP291). A portion of this research used resources at the High Flux Isotope Reactor, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory (IPTS‐27697, IPTS‐18982 and IPTS‐20263).

Keywords

Enzyme cluster
Enzyme immobilization
MOFs
SANS

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10.1002/anie.202522967

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title = "Monitoring Enzyme Clustering in a Highly Crystalline Metal–Organic Framework by Small-Angle Neutron Scattering",

abstract = "The molecular-level investigation of enzyme behavior in confined, cell-free environments is essential to understanding intrinsic properties and optimizing systems for desired functions. Metal–organic frameworks (MOFs) provide unique structural features that enable the immobilization of bulky biomolecules and allow direct probing of enzymatic behavior under confinement. Here, small-angle neutron scattering (SANS) was employed to probe porosity changes in a highly crystalline Tb-mesoMOF and to reveal the spatial arrangement of encapsulated enzymes across long-range length scales. Structural characteristics such as framework void space were resolved by SANS, while contrast-matching experiments using D2O/H2O mixtures suppressed background scattering from the MOF and isolated the enzyme contribution. Compared to unloaded Tb-mesoMOF, cytochrome c (Cyt. c)-loaded Tb-mesoMOF exhibited the emergence of a broad scattering feature at low q (∼0.005 {\AA}−1), indicative of enzyme clustering within the framework, accompanied by enhanced loading rate and capacity. Additional structural analyses using complementary techniques further corroborated these findings.",

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author = "Xiaoliang Wang and Lilin He and Shuo Qian and Shengyi Su and Farha, \{Omar K.\} and Shengqian Ma",

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AU - Wang, Xiaoliang

AU - He, Lilin

AU - Qian, Shuo

AU - Su, Shengyi

AU - Farha, Omar K.

AU - Ma, Shengqian

PY - 2026/1/22

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N2 - The molecular-level investigation of enzyme behavior in confined, cell-free environments is essential to understanding intrinsic properties and optimizing systems for desired functions. Metal–organic frameworks (MOFs) provide unique structural features that enable the immobilization of bulky biomolecules and allow direct probing of enzymatic behavior under confinement. Here, small-angle neutron scattering (SANS) was employed to probe porosity changes in a highly crystalline Tb-mesoMOF and to reveal the spatial arrangement of encapsulated enzymes across long-range length scales. Structural characteristics such as framework void space were resolved by SANS, while contrast-matching experiments using D2O/H2O mixtures suppressed background scattering from the MOF and isolated the enzyme contribution. Compared to unloaded Tb-mesoMOF, cytochrome c (Cyt. c)-loaded Tb-mesoMOF exhibited the emergence of a broad scattering feature at low q (∼0.005 Å−1), indicative of enzyme clustering within the framework, accompanied by enhanced loading rate and capacity. Additional structural analyses using complementary techniques further corroborated these findings.

AB - The molecular-level investigation of enzyme behavior in confined, cell-free environments is essential to understanding intrinsic properties and optimizing systems for desired functions. Metal–organic frameworks (MOFs) provide unique structural features that enable the immobilization of bulky biomolecules and allow direct probing of enzymatic behavior under confinement. Here, small-angle neutron scattering (SANS) was employed to probe porosity changes in a highly crystalline Tb-mesoMOF and to reveal the spatial arrangement of encapsulated enzymes across long-range length scales. Structural characteristics such as framework void space were resolved by SANS, while contrast-matching experiments using D2O/H2O mixtures suppressed background scattering from the MOF and isolated the enzyme contribution. Compared to unloaded Tb-mesoMOF, cytochrome c (Cyt. c)-loaded Tb-mesoMOF exhibited the emergence of a broad scattering feature at low q (∼0.005 Å−1), indicative of enzyme clustering within the framework, accompanied by enhanced loading rate and capacity. Additional structural analyses using complementary techniques further corroborated these findings.

KW - Enzyme cluster

KW - Enzyme immobilization

KW - MOFs

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JF - Angewandte Chemie - International Edition

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ER -

Monitoring Enzyme Clustering in a Highly Crystalline Metal–Organic Framework by Small-Angle Neutron Scattering

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