A REDOR ssNMR Investigation of the Role of an N‑Terminus Lysine in R5 Silica Recognition

Diatoms are unicellular algae that construct cell walls called frustules by the precipitation of silica, using special proteins that order the silica into a wide variety of nanostructures. The diatom species <i>Cylindrotheca fusiformis</i> contains proteins called silaffins within its frustules, which are believed to assemble into supramolecular matrices that serve as both accelerators and templates for silica deposition. Studying the properties of these biosilicification proteins has allowed the design of new protein and peptide systems that generate customizable silica nanostructures, with potential generalization to other mineral systems. It is essential to understand the mechanisms of aggregation of the protein and its coprecipitation with silica. We continue previous investigations into the peptide R5, derived from silaffin protein sil1p, shown to independently catalyze the precipitation of silica nanospheres in vitro. We used the solid-state NMR technique ¹³C­{²⁹Si} and ¹⁵N­{²⁹Si} REDOR to investigate the structure and interactions of R5 in complex with coprecipitated silica. These experiments are sensitive to the strength of magnetic dipole–dipole interactions between the ¹³C nuclei in R5 and the ²⁹Si nuclei in the silica and thus yield distance between parts of R5 and ²⁹Si in silica. Our data show strong interactions and short internuclear distances of 3.74 ± 0.20 Å between ¹³CO Lys3 and silica. On the other hand, the C_α and C_β nuclei show little or no interaction with ²⁹Si. This selective proximity between the K3 CO and the silica supports a previously proposed mechanism of rapid silicification of the antimicrobial peptide KSL (KKVVFKVKFK) through an imidate intermediate. This study reports for the first time a direct interaction between the N-terminus of R5 and silica, leading us to believe that the N-terminus of R5 is a key component in the molecular recognition process and a major factor in silica morphogenesis

Preparation of Ge@Organosilicon Core–Shell Structures and Characterization by Solid State NMR and Other Techniques

Many core–shell materials having a protecting outer layer have lately been proposed. In such materials it is not uncommon that chemical or thermal stability issues of the core material are resolved by a proper choice of the shell material. We report here the formation of core–shell structures by pyrolysis of a mixture of tetraethyl germanium and tetramethyl silicon at 750 °C in a simple one-step reaction without the use of catalysts under “RAPET” conditions. The composite product, germanium–core/organosilicon–shell (Ge@Organosilicon), is formed in two morphologies, rods and spheroids. The rods radial distribution is rather narrow while the spheroids exhibit a broader distribution due to their tendency to agglomerate. The germanium core phase is crystalline covered by a disordered organosilicon layer. The contribution of each of the precursors to the final product is shown by selected-area EDS and solid state NMR spectroscopy and further corroborated by RAMAN, EPR, and powder X-ray diffraction analysis

Studying the Conformation of a Silaffin-Derived Pentalysine Peptide Embedded in Bioinspired Silica using Solution and Dynamic Nuclear Polarization Magic-Angle Spinning NMR

Smart materials are created in nature at interfaces between biomolecules and solid materials. The ability to probe the structure of functional peptides that engineer biogenic materials at this heterogeneous setting can be facilitated tremendously by use of DNP-enhanced solid-state NMR spectroscopy. This sensitive NMR technique allows simple and quick measurements, often without the need for isotope enrichment. Here, it is used to characterize a pentalysine peptide, derived from a diatom’s silaffin protein. The peptide accelerates the formation of bioinspired silica and gets embedded inside the material as it is formed. Two-dimensional DNP MAS NMR of the silica-bound peptide and solution NMR of the free peptide are used to derive its secondary structure in the two states and to pinpoint some subtle conformational changes that the peptide undergoes in order to adapt to the silica environment. In addition, interactions between abundant lysine residues and silica surface are identified, and proximity of other side chains to silica and to neighboring peptide molecules is discussed

Design of Compact Biomimetic Cellulose Binding Peptides as Carriers for Cellulose Catalytic Degradation

The conversion of biomass into biofuels can reduce the strategic vulnerability of petroleum-based systems and at the same time have a positive effect on global climate issues. Lignocellulose is the cheapest and most abundant source of biomass and consequently has been widely considered as a source for liquid fuel. However, despite ongoing efforts, cellulosic biofuels are still far from commercial realization, one of the major bottlenecks being the hydrolysis of cellulose into simpler sugars. Inspired by the structural and functional modularity of cellulases used by many organisms for the breakdown of cellulose, we propose to mimic the cellulose binding domain (CBD) and the catalytic domain of these proteins by small molecular entities. Multiple copies of these mimics could subsequently be tethered together to enhance hydrolytic activity. In this work, we take the first step toward achieving this goal by applying computational approaches to the design of efficient, cost-effective mimetics of the CBD. The design is based on low molecular weight peptides that are amenable to large-scale production. We provide an optimized design of four short (i.e., ∼18 residues) peptide mimetics based on the three-dimensional structure of a known CBD and demonstrate that some of these peptides bind cellulose as well as or better than the full CBD. The structures of these peptides were studied by circular dichroism and their interactions with cellulose by solid phase NMR. Finally, we present a computational strategy for predicting CBD/peptide–cellulose binding free energies and demonstrate its ability to provide values in good agreement with experimental data. Using this computational model, we have also studied the dissociation pathway of the CBDs/peptides from the surface of cellulose

Changes to the Disordered Phase and Apatite Crystallite Morphology during Mineralization by an Acidic Mineral Binding Peptide from Osteonectin

Noncollagenous proteins regulate the formation of the mineral constituent in hard tissue. The mineral formed contains apatite crystals coated by a functional disordered calcium phosphate phase. Although the crystalline phase of bone mineral was extensively investigated, little is known about the disordered layer’s composition and structure, and less is known regarding the function of noncollagenous proteins in the context of this layer. In the current study, apatite was prepared with an acidic peptide (ON29) derived from the bone/dentin protein osteonectin. The mineral formed comprises needle-shaped hydroxyapatite crystals like in dentin and a stable disordered phase coating the apatitic crystals as shown using X-ray diffraction, transmission electron microscopy, and solid-state NMR techniques. The peptide, embedded between the mineral particles, reduces the overall phosphate content in the mineral formed as inferred from inductively coupled plasma and elemental analysis results. Magnetization transfers between disordered phase species and apatitic phase species are observed for the first time using 2D ¹H–³¹P heteronuclear correlation NMR measurements. The dynamics of phosphate magnetization transfers reveal that ON29 decreases significantly the amount of water molecules in the disordered phase and increases slightly their content at the ordered-disordered interface. The peptide decreases hydroxyl to disordered phosphate transfers within the surface layer but does not influence transfer within the bulk crystalline mineral. Overall, these results indicate that control of crystallite morphology and properties of the inorganic component in hard tissue by biomolecules is more involved than just direct interaction between protein functional groups and mineral crystal faces. Subtler mechanisms such as modulation of the disordered phase composition and structural changes at the ordered–disordered interface may be involved

Polyoxometalates entrapped in sol–gel matrices for reducing electron exchange column applications

<p>Electron exchange columns were developed by utilizing the redox properties of polyoxometalates (POMs) entrapped in silica matrices via the sol–gel route. The properties of the columns strongly depend on the composition of the precursors used to prepare the matrices. The columns exhibit good reversibility and are the first ‘reducing’ electron exchange columns ever prepared. They are also the first columns where both the matrix and the entrapped redox agent are inorganic compounds. This increases their stability. However, the redox properties of the entrapped POMs in the matrices are affected by the composition of the matrices.</p

Interfacial Mineral–Peptide Properties of a Mineral Binding Peptide from Osteonectin and Bone-like Apatite

Osteonectin is a regulator of bone mineralization. It interacts specifically with collagen and apatite through its N-terminal domain, inhibiting crystal growth. In this work, we investigated the interface formed between the mineral and an acidic peptide, ON29, derived from the protein’s apatite binding domain. The structural properties of the peptide bound to the mineral and the mineral–peptide interface are characterized using NMR and computational methods. A biomaterial complex is formed by precipitation of the mineral in the presence of the acidic peptide. The peptide gets embedded between mineral particles, which comprise a disordered hydrated coat covering apatite-like crystals. ³¹P SEDRA measurements show that the peptide does not affect the overall proximity between phosphate ions in the mineral. {¹⁵N}¹³C REDOR measurements reveal an α-turn in the center of the free peptide, which is unchanged when it is bound to the mineral. {³¹P}¹³C REDOR and ¹H–¹³C HETCOR measurements show that Glu/Asp carboxylates and Thr/Ala/Val side chains from ON29 are proximate to phosphate and hydroxyl groups in the mineral phases. Predictions of ON29’s fold on and off hydroxyapatite crystal faces using ROSETTA-surface are used to model the molecular conformation of the peptide and its apatite-binding interface. The models constructed without bias from experimental results are consistent with NMR measurements and map out extensively the residues forming an interface with apatitic crystals

Ammonia Treatment of 0.35Li₂MnO₃·0.65LiNi_0.35Mn_0.45Co_0.20O₂ Material: Insights from Solid-State NMR Analysis

Li-rich cathode materials of the formula <i>x</i>Li₂MnO₃·<i>y</i>LiNi_<i>a</i>Co_<i>b</i>Mn_<i>c</i>O₂ (<i>x</i> + <i>y</i> = 1, <i>a</i> + <i>b</i> + <i>c</i> = 1) boast very high discharge capacity, ca. 250 mAh/g. Yet, they suffer capacity decrease and average voltage fade during cycling in Li-ion batteries that prohibit their commercialization. Treatment of the materials with NH₃(g) at high temperatures produces improved electrodes with higher stability of capacity and average voltage. The present study follows the changes occurring in the materials upon treatment with ammonia gas, through ⁶Li and ⁷Li solid-state NMR investigations of the untreated and ammonia treated 0.35Li₂MnO₃·0.65LiNi_0.35Mn_0.45Co_0.20O₂ as well as its constituent phases, Li₂MnO₃ and LiNi_0.4Co_0.2Mn_0.4O₂. The NMR analysis demonstrates the biphasic nature of these materials. Furthermore, it shows that the Li₂MnO₃ component phase in the integrated material is the phase mostly being affected by the gas treatment. A thickening of a protective surface film in the integrated material, with the right exposure time to the reactive gas, is observed, which further precludes Ni leach out from the bulk and leads to improved electrode performance. Formation of minor electrochemically inactive oxide phases in the integrated material and similarly in the Li₂MnO₃ alone upon longer exposure to the gas suggests that the performance deterioration observed can be linked to the rearrangement of ions in the Li₂MnO₃ constituent phase in the integrated material

Thermodynamics of zinc binding to MamM-CTD dimer and mutants as measured by Isothermal Titration Calorimetry.

<p>Data was fitted to the single-site binding isotherm using ORIGIN 7.0 software.</p

Figure 3

<p>(A) ¹⁵N NMR spectra of [U-¹³C,¹⁵N] MamM-CTD, precipitated by zinc addition (blue) and the apo-protein precipitated using 2.2 M ammonium sulfate (red). Vertical dashed lines indicate imidazole resonances in the protein. (B) ¹³C NMR spectra of the same samples showing a shift in imidazole carbons (vertical dashed lines) and the presence of Trp247-C^γ carbon only in the zinc-precipitated protein (dotted line). (C-E) Slices of 2D ¹³C DARR spectrum of zinc-precipitated [U-¹³C, ¹⁵N] MamM-CTD with inter-subunit contacts shown in (C) and (E) and rigid zinc binding imidazole carbons indicated in (D) in accordance with spectrum in (B).</p