Maximizing the applicability of continuous wave (CW) Electron Paramagnetic Resonance (EPR): what more can we do after a century?

Pan, Yanxiong; Li, Qiaobin; Li, Hui; Lenertz, Mary; Jordahl, Drew; Suiter, Zoe; Chen, Bingcan; Yang, Zhongyu

doi:10.1016/j.jmro.2022.100060

Cited by 3 publications

(4 citation statements)

References 140 publications

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“…To investigate the molecular conformation of different formulations (L, PP-L, Z, PP-ZL), Fourier transform infrared (FTIR) spectroscopy was employed in the attenuated total reflection (ATR) mode. To probe the local backbone dynamics of the encapsulated lysozyme, electron paramagnetic resonance (EPR) spectroscopy was employed in the continuous wave (CW) mode . Details of these measurements are presented in the Supporting Information (SI).…”

Section: Methodsmentioning

confidence: 99%

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Metal–Organic Framework Induced Stabilization of Proteins in Polymeric Nanoparticles

Khan,

Armstrong,

Lenertz

et al. 2024

ACS Appl. Mater. Interfaces

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Developing protein confinement platforms is an attractive research area that not only promotes protein delivery but also can result in artificial environment mimicking of the cellular one, impacting both the controlled release of proteins and the fundamental protein biophysics. Polymeric nanoparticles (PNPs) are attractive platforms to confine proteins due to their superior biocompatibility, low cytotoxicity, and controllable release under external stimuli. However, loading proteins into PNPs can be challenging due to the potential protein structural perturbation upon contacting the interior of PNPs. In this work, we developed a novel approach to encapsulate proteins in PNPs with the assistance of the zeolitic imidazolate framework (ZIF). Here, ZIF offers an additional protection layer to the target protein by forming the protein@ZIF composite via aqueous-phase cocrystallization. We demonstrated our platform using a model protein, lysozyme, and a widely studied PNP composed of poly(ethylene glycol)poly(lactic-co-glycolic acid) (PEG−PLGA). A comprehensive study via standard loading and release tests as well as various spectroscopic techniques was carried out on lysozyme loaded onto PEG−PLGA with and without ZIF protection. As compared with the direct protein encapsulation, an additional layer with ZIF prior to loading offered enhanced loading capacity, reduced leaching, especially in the initial stage, led to slower release kinetics, and reduced secondary structural perturbation. Meanwhile, the function, cytotoxicity, and cellular uptake of proteins encapsulated within the ZIF-bound systems are decent. Our results demonstrated the use of ZIF in assisting in protein encapsulation in PNPs and established the basis for developing more sophisticated protein encapsulation platforms using a combination of materials of diverse molecular architectures and disciplines. As such, we anticipate that the protein-encapsulated ZIF systems will serve as future polymer protein confinement and delivery platforms for both fundamental biophysics and biochemistry research and biomedical applications where protein delivery is needed to support therapeutics and/or nutrients.

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Section: Methodsmentioning

confidence: 99%

“…To probe the local backbone dynamics of the encapsulated lysozyme, electron paramagnetic resonance (EPR) spectroscopy was employed in the continuous wave (CW) mode. 54 Details of these measurements are presented in the Supporting Information (SI).…”

Section: Protein Structural Characterizationmentioning

confidence: 99%

Metal–Organic Framework Induced Stabilization of Proteins in Polymeric Nanoparticles

Khan,

Armstrong,

Lenertz

et al. 2024

ACS Appl. Mater. Interfaces

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“…Doing so on multiple surface sites of the target enzyme can reveal the average motional restriction that an enzyme experiences in a MOF and areas of enzyme exposed above the surface of a MOF. 45 Meanwhile, the exposed area and backbone dynamics of enzymes are indicators of the MOF crystal property.…”

Section: ■ Introductionmentioning

confidence: 99%

“…We have also developed an experimental strategy to probe enzyme backbone dynamics in MOFs via site-directed spin labeling (SDSL)–electron paramagnetic resonance (EPR) spectroscopy and tested it in other MOFs. , SDSL–EPR probes enzyme dynamics and structural information at the residue level regardless of the complexities caused by the background and heterogeneity. − EPR is sensitive to protein backbone dynamics on the ∼ nanosecond order, , which can rapidly report if a labeled site is in contact with MOF crystal scaffolds or not; in the former case, EPR can also detect the subtle differences in enzyme backbone dynamics caused by differences in MOF crystallinity. , In a heterogeneous condition, EPR detects all contributions from different components; spectral simulation can then deconvolute the contribution of each. Doing so on multiple surface sites of the target enzyme can reveal the average motional restriction that an enzyme experiences in a MOF and areas of enzyme exposed above the surface of a MOF . Meanwhile, the exposed area and backbone dynamics of enzymes are indicators of the MOF crystal property.…”

Section: Introductionmentioning

confidence: 99%

Impact of Crystallinity on Enzyme Orientation and Dynamics upon Biomineralization in Metal–Organic Frameworks

Armstrong

MacRae

Lenertz

et al. 2023

ACS Appl. Mater. Interfaces

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Aqueous-phase co-crystallization (also known as biomimetic mineralization or biomineralization) is a unique way to encapsulate large enzymes, enzyme clusters, and enzymes with large substrates in metal–organic frameworks (MOFs), broadening the application of MOFs as enzyme carriers. The crystallinity of resultant enzyme@MOF biocomposites, however, can be low, raising a concern about how MOF crystal packing quality affects enzyme performance upon encapsulation. The challenges to overcome this concern are (1) the limited database of enzyme performance upon biomineralization in different aqueous MOFs and (2) the difficulty in probing enzyme restriction and motion in the resultant MOF scaffolds, which are related to the local crystal packing quality/density, under the interference of the MOF backgrounds. We have discovered several new aqueous MOFs for enzyme biomineralization with varied crystallinity [Expanding the Library of Metal–Organic Frameworks (MOFs) for Enzyme BiomineralizationJordahlD.ArmstrongZ.LiQ.GaoR.LiuW.JohnsonK.BrownW.ScheiwillerA.FengL.UgrinovA.MaoH.ChenB.QuadirM.PanY.LiH.YangZ. Jordahl, D. Armstrong, Z. Li, Q. Gao, R. Liu, W. Johnson, K. Brown, W. Scheiwiller, A. Feng, L. Ugrinov, A. Mao, H. Chen, B. Quadir, M. Pan, Y. Li, H. Yang, Z. ACS Appl. Mater. Interfaces2022145161951629]. Here, we address the second challenge by probing enzyme dynamics/restriction in these MOFs at the residue level via site-directed spin labeling (SDSL)–electron paramagnetic resonance (EPR) spectroscopy, a unique approach to determine protein backbone motions regardless of the background complexity. We encapsulated a model large-substrate enzyme, lysozyme, in eight newly discovered MOFs, which possess various degrees of crystallization, via aqueous-phase co-crystallization. Through the EPR study and simulations, we found rough connections between (a) enzyme mobility/dynamics and MOF crystal properties (packing quality and density) and (b) enzyme areas exposed above each MOF and their catalytic performance. This work suggests that protein SDSL and EPR can serve as an indicator of MOF crystal packing quality/density when biomineralized in MOFs. The method can be generalized to probing the dynamics of other enzymes on other solid surfaces/interfaces and guide the rational design of solid platforms (ca. MOF...

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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

Li,

Armstrong,

MacRae

et al. 2023

Chemical Physics Reviews

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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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Maximizing the applicability of continuous wave (CW) Electron Paramagnetic Resonance (EPR): what more can we do after a century?

Cited by 3 publications

References 140 publications

Metal–Organic Framework Induced Stabilization of Proteins in Polymeric Nanoparticles

Metal–Organic Framework Induced Stabilization of Proteins in Polymeric Nanoparticles

Impact of Crystallinity on Enzyme Orientation and Dynamics upon Biomineralization in Metal–Organic Frameworks

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

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