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Lysozyme is widely known to promote the formation of condensed silica networks from solutions containing silicic acid, in a reproducible and cost-effective way. However, little is known about the fate of the protein after the formation of the silica particles. Also, the relative arrangement of the different components in the resulting material is a matter of debate. In this study, we investigate the nature of the protein-silica interactions by means of solid-state nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and electron microscopy. We find that lysozyme and silica are in intimate contact and strongly interacting, but their interaction is neither covalent nor electrostatic: lysozyme is mostly trapped inside the silica by steric effects.

Paramagnetic NMR data contain extremely accurate long-range information on metalloprotein structures and, when used in the frame of integrative structural biology approaches, they allow for the retrieval of structural details to a resolution that is not achievable using other techniques. Paramagnetic data thus represent an extremely powerful tool to refine protein models in solution, especially when coupled to X-ray or cryoelectron microscopy data, to monitor the formation of complexes and determine the relative arrangements of their components, and to highlight the presence of conformational heterogeneity. More recently, theoretical and computational advancements in quantum chemical calculations of paramagnetic NMR observables are progressively opening new routes in structural biology, because they allow for the determination of the structure within the coordination sphere of the metal center, thus acting as a loupe on sites that are difficult to observe but very important for protein function.

Polycationic polypeptides prompt the polycondensation of inorganic oxides, most notably of silica. Hen egg-white lysozyme is a small polycationic protein that is quite conveniently used to this end. The fate of the protein after the completion of the polycondensation reaction is still a matter of debate. We have recently proven that lysozyme strongly interacts with silica. In this study, we use spin-label-based EPR spectroscopy to investigate whether the protein shows an orientational preference with respect to the silica surface within the composite. We find that a large share of the protein behaves as when it is adsorbed on pre-formed silica, albeit with a more marked preference for orientations that point the patches with higher surface charge density toward the material. In addition, a part of the protein shows a less-defined behavior. With this study, we provide additional information on the nature of the protein-material interactions in this class of bioinspired solids.

We present a processing method, based on the multivariate curve resolution approach (MCR), to denoise 2D solid-state NMR spectra, yielding a substantial S/N ratio increase while preserving the lineshapes and relative signal intensities. These spectral features are particularly important in the quantification of silicon species, where sensitivity is limited by the low natural abundance of the 29Si nuclei and by the dilution of the intrinsic protons of silica, but can be of interest also when dealing with other intermediate-to-low receptivity nuclei. This method also offers the possibility of coprocessing multiple 2D spectra that have the signals at the same frequencies but with different intensities (e.g.: as a result of a variation in the mixing time). The processing can be carried out on the time-domain data, thus preserving the possibility of applying further processing to the data. As a demonstration, we have applied Cadzow denoising on the MCR-processed FIDs, achieving a further increase in the S/N ratio and more effective denoising also on the transients at longer indirect evolution times. We have applied the combined denoising on a set of experimental data from a lysozyme-silica composite.

Biomacromolecules, such as proteins, often exhibit significant motions intimately associated with their function. Intrinsically disordered proteins and proteins with intrinsically disordered regions, although extremely important for a plethora of cellular functions, are difficult to structurally characterize at the atomic level because the experimental parameters report on ensemble and time averages. Here, we demonstrate for the C-terminal domain of the human immunodeficiency virus type 1 capsid protein that NMR and, in particular, residual dipolar couplings (RDCs) measured for the folded portion of the protein can inform on the structural preferences of the unstructured portion using RDC-prediction tools and the maximum occurrence approach.

Metalloproteins constitute a significant fraction of the proteome of all organisms and their characterization is critical for both basic sciences and biomedical applications. A large portion of metalloproteins bind paramagnetic metal ions, and paramagnetic NMR spectroscopy has been widely used in their structural characterization. However, the signals of nuclei in the immediate vicinity of the metal center are often broadened beyond detection. In this work, we show that it is possible to determine the coordination environment of the paramagnetic metal in the protein at a resolution inaccessible to other techniques. Taking the structure of a diamagnetic analogue as a starting point, a geometry optimization is carried out by fitting the pseudocontact shifts obtained from first principles quantum chemical calculations to the experimental ones.

Quantum chemical methods for calculating paramagnetic NMR observables are becoming increasingly accessible and are being included in the inorganic chemistry practice. Here, we test the performance of these methods in the prediction of proton hyperfine shifts of two archetypical high-spin pentacoordinate nickel(II) complexes (NiSAL-MeDPT and NiSAL-HDPT), which, for a variety of reasons, turned out to be perfectly suited to challenge the predictions to the finest level of detail. For NiSAL-MeDPT, new NMR experiments yield an assignment that perfectly matches the calculations. The slightly different hyperfine shifts from the two “halves” of the molecules related by a pseudo-C2 axis, which are experimentally divided into two well-defined spin systems, are also straightforwardly distinguished by the calculations. In the case of NiSAL-HDPT, for which no X-ray structure is available, the quality of the calculations allowed us to refine its structure using as a starting template the structure of NiSAL-MeDPT., State-of-the-art quantum chemical methods and paramagnetism-tailored NMR experiments provide a deep insight on the relation between the spectra and the electronic structure for two paramagnetic pentacoordinate nickel(II) complexes.

Nature has evolved several molecular machineries to promote the formation at physiological conditions of inorganic materials, which would otherwise be formed in extreme conditions. The molecular determinants of this process have been established over the last decade, identifying a strong role of electrostatics in the first steps of the precipitation. However, no conclusive, structure-based evidence has been provided so far. In this manuscript, we test the binding of lysozyme with silica and titania potential precursors. In contrast with the absence of structural information about the interaction with the silica precursor, we observe the interaction with a mononuclear titanium(IV) species, which is found to occur in a region rich of positive charges.

Hypothesis Amorphous Magnesium-Calcium Phosphate (AMCP) particles in the distal small intestine have been shown to have a fundamental role in mammals’ immune-surveillance mechanisms. Their formation in the gut lumen and their stability against crystallization are expected to depend upon physiological conditions such as pH and [Mg2+]. Knowing the influence of these parameters on AMCP stability would allow to predict the presence and the activity of the particles in physiological or pathological conditions. Experiments We performed the synthesis of AMCP particles at physiological temperature, in phosphate buffer at variable pH from ∼7.0 to 7.4. The stability of the particles was then tested by dispersing them in different conditions of [Mg2+], pH and concentration, so to mimic different biological conditions. The particles were characterized in terms of morphology, crystallinity, chemical composition and porosity. Findings The characterization showed that we managed to prepare AMCPs with features matching those of the endogenous particles. Both the lifetime of the amorphous phase and the nature of the formed crystalline material were found to depend upon [Mg2+], pH and concentration. This article paves the way for the comprehension of possible dysfunctions of the gut immune-surveillance mechanisms due to imbalances of these physico-chemical parameters.

Nuclear Magnetic Resonance is particularly sensitive to the electronic structure of matter and is thus a powerful tool to characterize in-depth the magnetic properties of a system. NMR is indeed increasingly recognized as an ideal tool to add precious structural information for the development of Single Ion Magnets, small complexes that are recently gaining much popularity due to their quantum computing and spintronics applications. In this review, we recall the theoretical principles of paramagnetic NMR, with particular attention to lanthanoids, and we give an overview of the recent advances in this field.

The properties of the conformational landscape of a biomolecule are of capital importance to understand its function. It is widely accepted that a statistical ensemble is far more representative than a single structure, especially for proteins with disordered regions. While experimental data provide the most important handle on the conformational variability that the system is experiencing, they usually report on either time or ensemble averages. Since the available conformations largely outnumber the (independent) available experimental data, the latter can be equally well reproduced by a variety of ensembles. We have proposed the Maximum Occurrence (MaxOcc) approach to provide an upper bound of the statistical weight of each conformation. This method is expected to converge towards the true statistical weights by increasing the number of independent experimental datasets. In this paper we explore the ability of DEER (Double Electron Electron Resonance) data, which report on the distance distribution between two spin labels attached to a biomolecule, to restrain the MaxOcc values and its complementarity to previously introduced experimental techniques such as NMR and Small-Angle X-ray Scattering. We here present the case of Ca2+ bound calmodulin (CaM) as a test case and show that DEER data impose a sizeable reduction of the conformational space described by high MaxOcc conformations.

Amorphous Magnesium-Calcium Phosphate (AMCP) particles in the distal small intestine have been shown to have a fundamental role in mammals' immune-surveillance mechanisms. Their formation in the gut lumen and their stability against crystallization are expected to depend upon physiological conditions such as pH and [MgWe performed the synthesis of AMCP particles at physiological temperature, in phosphate buffer at variable pH from ∼7.0 to 7.4. The stability of the particles was then tested by dispersing them in different conditions of [MgThe characterization showed that we managed to prepare AMCPs with features matching those of the endogenous particles. Both the lifetime of the amorphous phase and the nature of the formed crystalline material were found to depend upon [Mg