Role of Molecular Modification and Protein Folding in the Nucleation and Growth of Protein–Metal–Organic Frameworks

Abstract

Metal−organic frameworks (MOFs) are a class of porous nanomaterials that have been extensively studied as enzyme immobilization substrates. During in situ immobilization, MOF nucleation is driven by biomolecules with low isoelectric points. Investigation of how biomolecules control MOF self-assembly mechanisms on the molecular level is key to designing nanomaterials with desired physical and chemical properties. Here, we demonstrate how molecular modifications of bovine serum albumin (BSA) with fluorescein isothiocyanate (FITC) can affect MOF crystal size, morphology, and encapsulation efficiency. Final crystal properties are characterized using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), fluorescent microscopy, and fluorescence spectroscopy. To probe MOF self-assembly, in situ experiments were performed using cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction (XRD). Biophysical characterization of BSA and FITC-BSA was performed using ζ potential, mass spectrometry, circular dichroism studies, fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The combined data reveal that protein folding and stability within amorphous precursors are contributing factors in the rate, extent, and mechanism of crystallization. Thus, our results suggest molecular modifications as promising methods for fine-tuning protein@ MOFs' nucleation and growth.

The role of protein folding in prenucleation clusters on the activity of enzyme@metal–organic frameworks

“…Moreover, biomolecular unfolding from precursor interactions can additionally affect the formation mechanism and final MOF structure, which has been demonstrated in an FITC-BSA@ZIF-8 model. 17,32 Furthermore, while the amorphous structure that we report in 4 : 1 GOx@ZIF-8 aligns with some reports that have also observed amorphous ZIF-8 at this condition, 8 other studies have also reported crystalline sod products at the same L : M ratio. 6 We suggest that these differences may be due to the differing stabilities, structures, and purities of GOx obtained by different laboratories.…”

“…In previous research, we have shown that biomolecule folding affects the crystallization mechanism, kinetics, and structural properties of MOFs synthesized in situ. 17 This study also found that the MOF precursors, specically zinc acetate, can inuence the initial protein folding. Other studies have shown that hydrophilic ligands can stabilize enzymes in their active conformation, retaining activity of the enzyme in the nal enzyme@MOF, 18 whereas more hydrophobic ligands lead to a loss of functionality.…”

“…21,22 Instead, we used CD spectroscopy to examine enzyme folding in the presence of divalent zinc (Zn), as previous studies have found that proteins can form stable complexes with each of the precursors. 17 CD spectroscopy shows that GOx/Zn solutions exhibit a slight reduction in alpha helical character as compared to GOx without Zn (Fig. 3a).…”

Understanding and controlling the nucleation and growth of metal–organic frameworks

“…114 With this insight, molecular modifications of proteins have shown to be promising methods for tailoring the surface chemistry to promote nucleation and growth of a particular MOF phase and morphology. 122 Furthermore, proteins are believed to add to the growing crystal through particle addition. Through addition, the proteins create defects in the crystal as they are too complex to match the lattice planes of the MOF.…”

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization

“…Different chemical compositions may lead to different degrees of enzyme coating by the metal crystals. In fact, recently, researchers have shown how protein folding and molecular modification define the rate, extent, and mechanism of crystallization when mixed with 2-methylimidazole and zinc acetate [ 59 ]. We can assume that these effects may be also produced when mineralizing immobilized enzymes, and that they can produce different effects on the enzyme properties.…”