Spatially Confined Protein Assembly in Hierarchical Mesoporous Metal-Organic Framework

To gain insights into the spatial arrangement of biomolecules within the nanopores, the researchers used in situ small-angle neutron scattering (SANS) to probe deuterated green fluorescent protein (d-GFP) entrapped in a mesoporous MOF.
Spatially Confined Protein Assembly in Hierarchical Mesoporous Metal-Organic Framework
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    The immobilization of biomolecules into porous materials played a crucial role in industrialization, leading to improved stability and easier separation for reuse. Despite significant advances made in recent years through indirect methods, our understanding of the structural conformation and spatial arrangement of these biomolecules remains limited. Direct monitoring of their conformations is challenging, but gaining a better understanding of their behavior in confined environments could help in designing and tailoring the properties for various applications. To this end, we employed in situ small-angle neutron scattering (SANS) to study the spatial arrangement of green fluorescent protein (GFP) trapped in a mesoporous matrix. With its ability to detect length scales from 1 to 200 nm, SANS is well-suited for studying the structures of biomolecules in mesoporous materials. It can minimize scattering contributions from the solid matrices by adjusting the scattering contrast of solvent with the variation of deuterated and hydrogenated solvents (H2O and D2O), which allows us to extract structural parameters and determine the overall structure of biomacromolecules in a multi-component system. The scattering intensity is proportional to the square of the Fourier Transform of the NSLD distribution, averaged over all orientations, and the azimuthally averaged 1D curve against the scattering angle (or q-vector) provides information on molecular weights, dimensions, and low-resolution shapes. Metal-Organic Frameworks (MOFs) are selected as the matric support, due to their unique structural features resulting from the assembly of a wide range of metal ion/metal ion cluster nodes and multitopic organic ligands. Compared to traditional porous materials, MOFs have a large pore size, adjustable pore structure, and high surface area, which combine to give them exceptional loading capacity for biomolecules. Usually, the measurement of protein structures is conducted using conventional spectroscopic analysis, such as solid-state UV−visible spectrophotometry, Raman and FTIR. However, these phenomenal techniques generally give indirect evidence to probe the confinement-induced conformational changes or dynamic constraints due to the difficulty of spectral assessment and complexity of protein-protein or protein-matrix interactions, and they are difficult to provide the direct depiction of their arrangement or overall structure under confinement environment.

    Employing the SANS technique, we were able to for the first time determine the protein spatial arrangement within the MOF nanopores as illustrated in the present work.  Considering the low scattering contrast between h-GFP and backbone of MOF-919, deuterated GFP (d-GFP) with a much higher contrast matching point than h-GFP, was used in this work. The ubiquitous existence of hydrogen in soft matter and biomolecules enables the utilization of isotope deuterium to enhance the contrast, by replacing H with D during the protein expression without altering their structures and chemical properties. To investigate the effects of protein concentration on its distribution inside hierarchical MOF-919, a variety of d-GFP@MOF-919 composites (denoted as C1-C4) were prepared via loading in decreasing concentration of d-GFP in 20 mM Tris-buffer (pH 7.5, 50 mM NaCl), respectively, under ambient conditions.  Going further in visualization of protein arrangement confined in the hierarchical structure of MOF-919, all d-GFP@MOF-919s were reconstructed with ab initio method implemented in DAMMIF, a data analysis package for small angle scattering data. Based on the reconstructions and the maximum dimension in P(r) analysis, the size of GFP particles in C1 and C2 samples is much larger than GFP monomer, likely an oligomer or a cluster GFP encaged (or constrained) by the MOF lattice. However, C1 is more likely to form larger group like “tetramers” among MOF cavities and tends to cover four adjacent cages via the reconstruction. C3 and C4 exhibit more similar arrangement to each other, and the reconstruction reveals a GFP assembly in an interesting configuration, resembling a GFP monomer perpendicular to another GFP due to the MOF constraint in geometry.  It is noted that, with the increasing loading of GFP in MOF structure, all arrangements have a peak (or hump) representing the GFP assembly around 32-42 Å, while the most probable pair distance of the free d-GFP calculated in solution is peaked at around 25-30 Å. This indicates that the protein assembly is different with in solution due to the confined environment of MOF and it is believed that the existence of protein-protein interactions and confinement effects synergistically resulted in the closer protein arrangement.

    This work showed that SANS can directly visualize proteins encapsulated within the nanopores of MOFs. The contrast matching method, along with d-GFP, produced a strong scattering signal, allowing for the distinction of the protein component from the hydrogenated MOF at the contrast matching point. The scattering profiles at high q range revealed the protein assembly inside the pores. The presence of numerous large openings surrounding cages and connecting two mesopores is believed to facilitate the formation of protein "assemblies", especially through the cage apertures of MOF-919. This is the first time that the protein spatial arrangement in MOFs has been revealed through SANS, and the use of fully deuterated protein resulted in higher neutron scattering, overcoming the limitation of low scattering contrast between the MOF and protonated protein. The highly ordered structure features of the matrix MOF were clearly shown by SANS, enabling the easy characterization and modeling of the overall protein structure. The unique feature of contrast matching allows for effective extraction of structure information of confined proteins in-situ, selectively from the host matrix MOF-919. This is a crucial attempt to develop a novel and simple approach for probing the behavior of proteins and other biomolecules in the nanospace of MOFs, and it will be significant for applications in biocatalysis, biomedicine, and beyond.

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Go to the profile of Xiangke Wang
3 months ago

A very excellent research.