Branched Polymeric Prenucleation Assemblies Initiate Calcium Phosphate Precipitation.

The formation of crystalline calcium phosphate (CaP) has recently gained ample attention as it does not follow the classic nucleation-and-growth mechanism of solid formation. Instead, the precipitation mechanisms can involve numerous intermediates, including soluble prenucleation species. However, structural features, stability, and transformation of such solution-state precursors remain largely undisclosed. Herein, we report a detailed and comprehensive characterization of the sequential events involved in calcium phosphate crystallization starting from the very early prenucleation stage. We integrated an extensive set of time-resolved methods, including NMR, turbidimetry, SAXS, cryo-TEM, and calcium-potentiometry to show that CaP nucleation is initiated by the transformation of "branched" polymeric prenucleation assemblies into amorphous calcium phosphate spheres. Such a mineralization process starts with the spontaneous formation of so-called nanometric prenucleation clusters (PNCs) that later assemble into those branched polymeric assemblies without calcium ion uptake from the solution. Importantly, the branched macromolecular species are invisible to many techniques (NMR, turbidity, calcium-potentiometry) but can readily be evidenced by time-resolved SAXS. We find that these polymeric assemblies constitute the origin of amorphous calcium phosphate (ACP) precipitation through an unexpected process: spontaneous dissolution is followed by local densification of 100-200 nm wide domains leading to ACP spheres of similar size. Finally, we demonstrate that the timing of the successive events involved in the CaP mineralization pathway can be kinetically controlled by the Ca2+/Pi molar ratio, such that the lifetime of the soluble transient species can be increased up to hours when decreasing it.

Hyperpolarized nuclear Overhauser enhancement of alanine methyl groups by doubly relayed proton exchange.

Hyperpolarized water in dissolution dynamic nuclear polarization (dDNP) experiments has emerged as a promising method for enhancing nuclear magnetic resonance (NMR) signals, particularly in studies of proteins and peptides. Herein, we focus on the application of "proton exchange-doubly relayed" nuclear Overhauser effects (NOE) from hyperpolarized water to achieve positive signal enhancement of methyl groups in the side chain of an alanine-glycine peptide. In particular, we show a cascade hyperpolarization transfer. Initial proton exchange between solvent and amide introduces hyperpolarization into the peptide. Subsequently, intermolecular NOE relays the hyperpolarization first to Ala-Hα and then in a second step to the Ala-CH3 moiety. Both NOEs have negative signs. Hence, the twice-relayed NOE pathway leads to a positive signal enhancement of the methyl group with respect to the thermal equilibrium magnetization. This effect might indicate a way towards hyperpolarized water-based signal enhancement for methyl groups, which are often used for NMR studies of large proteins in solution.

An Atomistic View on the Mechanism of Diatom Peptide-Guided Biomimetic Silica Formation.

Deciphering nature's remarkable way of encoding functions in its biominerals holds the potential to enable the rational development of nature-inspired materials with tailored properties. However, the complex processes that convert solution-state precursors into solid biomaterials remain largely unknown. In this study, an unconventional approach is presented to characterize these precursors for the diatom-derived peptides R5 and synthetic Silaffin-1A1 (synSil-1A1). These molecules can form defined supramolecular assemblies in solution, which act as templates for solid silica structures. Using a tailored structural biology toolbox, the structure-function relationships of these self-assemblies are unveiled. NMR-derived constraints are employed to enable a recently developed fractal-cluster formalism and then reveal the architecture of the peptide assemblies in atomistic detail. Finally, by monitoring the self-assembly activities during silica formation at simultaneous high temporal and residue resolution using real-time spectroscopy, the mechanism is elucidated underlying template-driven silica formation. Thus, it is demonstrated how to exercise morphology control over bioinorganic solids by manipulating the template architectures. It is found that the morphology of the templates is translated into the shape of bioinorganic particles via a mechanism that includes silica nucleation on the solution-state complexes' surfaces followed by complete surface coating and particle precipitation.

NMR-identification of the interaction between BRCA1 and the intrinsically disordered monomer of the Myc-associated factor X.

The breast cancer susceptibility 1 (BRCA1) protein plays a pivotal role in modulating the transcriptional activity of the vital intrinsically disordered transcription factor MYC. In this regard, mutations of BRCA1 and interruption of its regulatory activity are related to hereditary breast and ovarian cancer (HBOC). Interestingly, so far, MYC's main dimerization partner MAX (MYC-associated factor X) has not been found to bind BRCA1 despite a high sequence similarity between both oncoproteins. Herein, we show that a potential reason for this discrepancy is the heterogeneous conformational space of MAX, which encloses a well-documented folded coiled-coil homodimer as well as a less common intrinsically disordered monomer state-contrary to MYC, which exists mostly as intrinsically disordered protein in the absence of any binding partner. We show that when the intrinsically disordered state of MAX is artificially overpopulated, the binding of MAX to BRCA1 can readily be observed. We characterize this interaction by nuclear magnetic resonance (NMR) spectroscopy chemical shift and relaxation measurements, complemented with ITC and SAXS data. Our results suggest that BRCA1 directly binds the MAX monomer to form a disordered complex. Though probed herein under biomimetic in-vitro conditions, this finding can potentially stimulate new perspectives on the regulatory network around BRCA1 and its involvement in MYC:MAX regulation.

Biphasic NMR of Hyperpolarized Suspensions-Real-Time Monitoring of Solute-to-Solid Conversion to Watch Materials Grow.

Nuclear magnetic resonance (NMR) spectroscopy is a key method for the determination of molecular structures. Due to its intrinsically high (i.e., atomistic) resolution and versatility, it has found numerous applications for investigating gases, liquids, and solids. However, liquid-state NMR has found little application for suspensions of solid particles as the resonances of such systems are excessively broadened, typically beyond the detection threshold. Herein, we propose a route to overcoming this critical limitation by enhancing the signals of particle suspensions by >3.000-fold using dissolution dynamic nuclear polarization (d-DNP) coupled with rapid solid precipitation. For the proof-of-concept series of experiments, we employed calcium phosphate (CaP) as a model system. By d-DNP, we boosted the signals of phosphate 31P spins before rapid CaP precipitation inside the NMR spectrometer, leading to the inclusion of the hyperpolarized phosphate into CaP-nucleated solid particles within milliseconds. With our approach, within only 1 s of acquisition time, we obtained spectra of biphasic systems, i.e., micrometer-sized dilute solid CaP particles coexisting with their solution-state precursors. Thus, this work is a step toward real-time characterization of the solid-solution equilibrium. Finally, integrating the hyperpolarized data with molecular dynamics simulations and electron microscopy enabled us to shed light on the CaP formation mechanism in atomistic detail.