28• Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration 29 stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called 30 silica cells deposit silica in most of their volume by unknown mechanism. 31• Using bioinformatics tools, we identified a previously uncharacterized protein in 32 sorghum (Sorghum bicolor), which we named Siliplant1 (Slp1). Silica precipitation 33 activity in vitro, expression profile, and activity in precipitating biosilica in vivo were 34 characterized. 35• Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. 36 A short peptide, repeating five times in the protein precipitated silica in vitro at a 37 biologically relevant silicic acid concentration. Raman and NMR spectroscopies 38 showed that the peptide attached the silica through lysine amine groups, forming a 39 mineral-peptide open structure. We found Slp1 expression in immature leaf and 40 inflorescence tissues. In the immature leaf active silicification zone, Slp1 was localized 41 to the cytoplasm or near cell boundaries of silica cells. It was packed in vesicles and 42 secreted to the paramural space. Transient overexpression of Slp1 in sorghum resulted 43 in ectopic silica deposition in all leaf epidermal cell types. 44 • Our results show that Slp1 precipitates silica in sorghum silica cells. 45 46 47
Deriving the conformation of adsorbed proteins is important in the assessment of their functional activity when immobilized. This has particularly important bearings on the design of contemporary and new encapsulated enzyme-based drugs, biosensors, and other bioanalytical devices. Solid-state nuclear magnetic resonance (NMR) measurements can expand our molecular view of proteins in this state and of the molecular interactions governing protein immobilization on popular biocompatible surfaces such as silica. Here, the authors study the immobilization of ubiquitin on the mesoporous silica MCM41 by NMR and other techniques. Protein molecules are shown to bind efficiently at pH 5 through electrostatic interactions to individual MCM41 particles, causing their agglutination. The strong attraction of ubiquitin to MCM41 surface is given molecular context through evidence of proximity of basic, carbonyl and polar groups on the protein to groups on the silica surface using NMR measurements. The immobilized protein exhibits broad peaks in two-dimensional C dipolar-assisted rotational resonance spectra, an indication of structural multiplicity. At the same time, cross-peaks related to Tyr and Phe sidechains are missing due to motional averaging. Overall, the favorable adsorption of ubiquitin to MCM41 is accompanied by conformational heterogeneity and by a major loss of motional degrees of freedom as inferred from the marked entropy decrease. Nevertheless, local motions of the aromatic rings are retained in the immobilized state.
Protein immobilization on material surfaces is emerging as a powerful tool in the design of devices and active materials for biomedical and pharmaceutical applications as well as for catalysis. Preservation of the protein’s biological functionality is crucial to the design process and is dependent on the ability to maintain its structural and dynamical integrity while removed from the natural surroundings. The scientific techniques to validate the structure of immobilized proteins are scarce and usually provide limited information as a result of poor resolution. In this work, we benchmarked the ability of standard solid-state NMR techniques to resolve the effects of binding to dissimilar silica materials on a model protein. In particular, the interactions between ubiquitin and the surfaces of MCM41, SBA15, and silica formed in situ were tested for their influence on the structure and dynamics of the protein. It is shown that the protein’s globular fold in the free state is only slightly perturbed in the three silica materials. Local motions on a residue level that are quenched by immobilization or, conversely, that arise from the process are also detailed. NMR measurements show that these perturbations are unique to each silica material and can serve as reporters of the characteristic surface chemistry.
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