Solid-state NMR Approach Research Articles

Monitoring and understanding the paraelectric-ferroelectric phase transition in the metal-organic framework [NH4 ][M(HCOO)3 ] by solid-state NMR spectroscopy.

The paraelectric-ferroelectric phase transition in two isostructural metal-organic frameworks (MOFs) [NH4 ][M(HCOO)3 ] (M=Mg, Zn) was investigated by in situ variable-temperature (25) Mg, (67) Zn, (14) N, and (13) C solid-state NMR (SSNMR) spectroscopy. With decreasing temperature, a disorder-order transition of NH4 (+) cations causes a change in dielectric properties. It is thought that [NH4 ][Mg(HCOO)3 ] exhibits a higher transition temperature than [NH4 ][Zn(HCOO)3 ] due to stronger hydrogen-bonding interactions between NH4 (+) ions and framework oxygen atoms. (25) Mg and (67) Zn NMR parameters are very sensitive to temperature-induced changes in structure, dynamics, and dielectric behavior; stark spectral differences across the paraelectric-ferroelectric phase transition are intimately related to subtle changes in the local environment of the metal center. Although (25) Mg and (67) Zn are challenging nuclei for SSNMR experiments, the highly spherically symmetric metal-atom environments in [NH4 ][M(HCOO)3 ] give rise to relatively narrow spectra that can be acquired in 30-60 min at a low magnetic field of 9.4 T. Complementary (14) N and (13) C SSNMR experiments were performed to probe the role of NH4 (+) -framework hydrogen bonding in the paraelectric-ferroelectric phase transition. This multinuclear SSNMR approach yields new physical insights into the [NH4 ][M(HCOO)3 ] system and shows great potential for molecular-level studies on electric phenomena in a wide variety of MOFs.

Probing the Impact of Acidification on Spider Silk Assembly Kinetics.

Spiders utilize fine adjustment of the physicochemical conditions within its silk spinning system to regulate spidroin assembly into solid silk fibers with outstanding mechanical properties. However, the exact mechanism about which this occurs remains elusive and is still hotly debated. In this study, the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. Incubating the protein-rich MA silk gland fluid at acidic pH conditions results in the formation of silk fibers that are 10-100 μm in length and ∼2 μm in diameter as judged by optical and electron microscope methods. The in vitro spider silk assembly kinetics were monitored as a function of pH with a (13)C solid-state MAS NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation as well as the percentage of β-sheet structure in the grown fibers depend on the pH. These results confirm the importance of an acidic pH gradient along the spinning duct for spider silk formation and provide a powerful spectroscopic approach to probe the kinetics of spider silk formation under various biochemical conditions.

Dynamics-based selective 2D (1)H/(1)H chemical shift correlation spectroscopy under ultrafast MAS conditions.

Dynamics plays important roles in determining the physical, chemical, and functional properties of a variety of chemical and biological materials. However, a material (such as a polymer) generally has mobile and rigid regions in order to have high strength and toughness at the same time. Therefore, it is difficult to measure the role of mobile phase without being affected by the rigid components. Herein, we propose a highly sensitive solid-state NMR approach that utilizes a dipolar-coupling based filter (composed of 12 equally spaced 90° RF pulses) to selectively measure the correlation of (1)H chemical shifts from the mobile regions of a material. It is interesting to find that the rotor-synchronized dipolar filter strength decreases with increasing inter-pulse delay between the 90° pulses, whereas the dipolar filter strength increases with increasing inter-pulse delay under static conditions. In this study, we also demonstrate the unique advantages of proton-detection under ultrafast magic-angle-spinning conditions to enhance the spectral resolution and sensitivity for studies on small molecules as well as multi-phase polymers. Our results further demonstrate the use of finite-pulse radio-frequency driven recoupling pulse sequence to efficiently recouple weak proton-proton dipolar couplings in the dynamic regions of a molecule and to facilitate the fast acquisition of (1)H/(1)H correlation spectrum compared to the traditional 2D NOESY (Nuclear Overhauser effect spectroscopy) experiment. We believe that the proposed approach is beneficial to study mobile components in multi-phase systems, such as block copolymers, polymer blends, nanocomposites, heterogeneous amyloid mixture of oligomers and fibers, and other materials.

Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition

The genomics and proteomics revolutions have been enormously successful in providing crucial “parts lists” for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The “sum-of-the-parts” bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture.

Probing the Huntingtin 1-17 Membrane Anchor on a Phospholipid Bilayer by Using All-Atom Simulations

Mislocalization and aggregation of the huntingtin protein are related to Huntington’s disease. Its first exon—more specifically the first 17 amino acids (Htt17)—is crucial for the physiological and pathological functions of huntingtin. It regulates huntingtin’s activity through posttranslational modifications and serves as an anchor to membrane-containing organelles of the cell. Recently, structure and orientation of the Htt17 membrane anchor were determined using a combined solution and solid-state NMR approach. This prompted us to refine this model by investigating the dynamics and thermodynamics of this membrane anchor on a POPC bilayer using all-atom, explicit solvent molecular dynamics and Hamiltonian replica exchange. Our simulations are combined with various experimental measurements to generate a high-resolution atomistic model for the huntingtin Htt17 membrane anchor on a POPC bilayer. More precisely, we observe that the single α-helix structure is more stable in the phospholipid membrane than the NMR model obtained in the presence of dodecylphosphocholine detergent micelles. The resulting Htt17 monomer has its hydrophobic plane oriented parallel to the bilayer surface. Our results further unveil the key residues interacting with the membrane in terms of hydrogen bonds, salt-bridges, and nonpolar contributions. We also observe that Htt17 equilibrates at a well-defined insertion depth and that it perturbs the physical properties—order parameter, thickness, and area per lipid—of the bilayer in a manner that could favor its dimerization. Overall, our observations reinforce and refine the NMR measurements on the Htt17 membrane anchor segment of huntingtin that is of fundamental importance to its biological functions.

A New Understanding of Antibiotic Action via Solid-State NMR of Cells with Uniform Isotopic Labeling

In this issue of the Biophysical Journal, Nygaard et al. (1) describe an exciting new solid-state NMR approach to probe the composition of whole bacterial cells and their cell-wall fractions. Use of uniform 13C and 15N labeling as well as magic-angle spinning and rotational-echo double-resonance (REDOR) selection of either Gly or non-Gly 13C signals allows for NMR detection of many chemical functionalities including polysaccharide and protein (2–5). In my view, the most exciting result is the striking differences among unfiltered 13C NMR spectra of whole bacterial cells grown with different antibiotics, as displayed in Fig. 7 in the article.

Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR.

Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. (19)F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively (19)F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. (31)P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, (2)H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.

Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach.

The β-barrel assembly machinery (BAM) is involved in folding and insertion of outer membrane proteins in Gram-negative bacteria, a process that is still poorly understood. With its 790 residues, BamA presents a challenge to current NMR methods. We utilized a "divide and conquer" approach in which we first obtained resonance assignments for BamA's periplasmic POTRA domains 4 and 5 by solution NMR. Comparison of these assignments to solid-state NMR (ssNMR) data obtained on two BamA constructs including the transmembrane domain and one or two soluble POTRA domains suggested that the fold of POTRA domain 5 critically depends on the interface with POTRA 4. Using specific labeling schemes we furthermore obtained ssNMR resonance assignments for residues in the extracellular loop 6 that is known to be crucial for BamA-mediated substrate folding and insertion. Taken together, our data provide novel insights into the conformational stability of membrane-embedded, non-crystalline BamA.

Nano-mole scale sequential signal assignment by (1)H-detected protein solid-state NMR.

We present a 3D (1)H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10-100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at ∼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers ∼110 fold time saving over a traditional 3D (13)C-detected SSNMR approach.

A multinuclear solid state NMR spectroscopic study of the structural evolution of disordered calcium silicate sol-gel biomaterials.

Disordered sol-gel prepared calcium silicate biomaterials show significant, composition dependent ability to bond with bone. Bone bonding is attributed to rapid hydroxycarbonate apatite (HCA) formation on the glass surface after immersion in body fluid (or implantation). Atomic scale details of the development of the structure of (CaO)x(SiO2)1-x (x = 0.2, 0.3 and 0.5) under heat treatment and subsequent dissolution in simulated body fluid (SBF) are revealed through a multinuclear solid state NMR approach using one-dimensional (17)O, (29)Si, (31)P and (1)H. Central to this study is the combination of conventional static and magic angle spinning (MAS) and two-dimensional (2D) triple quantum (3Q) (17)O NMR experiments that can readily distinguish and quantify the bridging (BOs) and non-bridging (NBOs) oxygens in the silicate network. Although soluble calcium is present in the sol, the (17)O NMR results reveal that the sol-gel produced network structure is initially dominated by BOs after gelation, aging and drying (e.g. at 120 °C), indicating a nanoscale mixture of the calcium salt and a predominantly silicate network. Only once the calcium salt is decomposed at elevated temperatures do the Ca(2+) ions become available to break BO. Apatite forming ability in SBF depends strongly on the surface OH and calcium content. The presence of calcium aids HCA formation via promotion of surface hydration and the ready availability of Ca(2+) ions. (17)O NMR shows the rapid loss of NBOs charge balanced by calcium as it is leached into the SBF. The formation of nanocrystalline, partially ordered HCA can be detected via(31)P NMR. This data indicates the importance of achieving the right balance of BO/NBO for optimal biochemical response and network properties.

Probing the nanostructure, interfacial interaction, and dynamics of chitosan-based nanoparticles by multiscale solid-state NMR.

Chitosan-based nanoparticles (NPs) are widely used in drug and gene delivery, therapy, and medical imaging, but a molecular-level understanding of the internal morphology and nanostructure size, interface, and dynamics, which is critical for building fundamental knowledge for the precise design and efficient biological application of the NPs, remains a great challenge. Therefore, the availability of a multiscale (0.1-100 nm) and nondestructive analytical technique for examining such NPs is of great importance for nanotechnology. Herein, we present a new multiscale solid-state NMR approach to achieve this goal for the investigation of chitosan-poly(N-3-acrylamidophenylboronic acid) NPs. First, a recently developed (13)C multiple cross-polarization magic-angle spinning (MAS) method enabled fast quantitative determination of the NPs' composition and detection of conformational changes in chitosan. Then, using an improved (1)H spin-diffusion method with (13)C detection and theoretical simulations, the internal morphology and nanostructure size were quantitatively determined. The interfacial coordinated interaction between chitosan and phenylboronic acid was revealed by one-dimensional MAS and two-dimensional (2D) triple-quantum MAS (11)B NMR. Finally, dynamic-editing (13)C MAS and 2D (13)C-(1)H wide-line separation experiments provided details regarding the componential dynamics of the NPs in the solid and swollen states. On the basis of these NMR results, a model of the unique nanostructure, interfacial interaction, and componential dynamics of the NPs was proposed.

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can provide 3D structural information. However, there are numerous challenges to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges in order to obtain high-resolution structural insights into electron transfer processes mediated by membrane-bound proteins like mammalian cytochrome-b5, cytochrome-P450 and cytochrome-P450-reductase. In this study, we demonstrate the feasibility of using dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement. Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrate the feasibility of suppressing the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

Characterization of the Vibrio cholerae extracellular matrix: A top-down solid-state NMR approach

Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using 13C CPMAS and 13C{15N}, 15N{31P}, and 13C{31P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, 15N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

ChemInform Abstract: Recent Advances in Solid‐State Nuclear Magnetic Resonance Techniques to Quantify Biomolecular Dynamics

AbstractReview: 52 refs.

Understanding the aggregation of bis(benzoxazolyl)stilbene in PLA/PBS blends: a combined spectrofluorimetric, calorimetric and solid state NMR approach

Poly(lactic acid)/poly(butylene succinate) (PLA/PBS) blends, containing a small amount of 4,4′-bis(2-benzoxazolyl)stilbene (BBS) dye, showed a chromogenic response after heating above 80 °C, due to the formation of BBS aggregates. This smart behaviour can be useful for many different applications, such as for the realization of time–temperature optical indicators, and the understanding of the processes lying behind the aggregation of chromophores is fundamental for improving their performances. In this work, the phase transitions occurring in the PLA/PBS polymeric matrix under heating were investigated in detail by combining spectrofluorimetric, DSC, and variable-temperature solid state NMR (SSNMR) measurements on both PLA- (in pellets and film) and PLA/PBS-BBS films. A detailed characterization of the phase behaviour of both PLA and PBS domains was achieved. More specifically, the SSNMR analysis of 1H FID at different temperatures was found to be a very powerful and useful approach to investigate the phase transitions of PLA, complementary to DSC. Different phase and dynamic processes, PBS and PLA cold crystallization and PLA glass transition, seem to concur, at different levels, to promote BBS aggregation. A possible mechanism for the formation of BBS aggregates under heating was proposed.

Probing Hydrogen Bond Networks in Half-Sandwich Ru(II) Building Blocks by a Combined 1H DQ CRAMPS Solid-State NMR, XRPD, and DFT Approach

The hydrogen bond network of three polymorphs (1α, 1β, and 1γ) and one solvate form (1·H2O) arising from the hydration-dehydration process of the Ru(II) complex [(p-cymene)Ru(κN-INA)Cl2] (where INA is isonicotinic acid), has been ascertained by means of one-dimensional (1D) and two-dimensional (2D) double quantum (1)H CRAMPS (Combined Rotation and Multiple Pulses Sequences) and (13)C CPMAS solid-state NMR experiments. The resolution improvement provided by homonuclear decoupling pulse sequences, with respect to fast MAS experiments, has been highlighted. The solid-state structure of 1γ has been fully characterized by combining X-ray powder diffraction (XRPD), solid-state NMR, and periodic plane-wave first-principles calculations. None of the forms show the expected supramolecular cyclic dimerization of the carboxylic functions of INA, because of the presence of Cl atoms as strong hydrogen bond (HB) acceptors. The hydration-dehydration process of the complex has been discussed in terms of structure and HB rearrangements.

Monitoring the Electrochemical Processes in the Lithium-Air Battery by Solid State NMR Spectroscopy.

A multi-nuclear solid-state NMR approach is employed to investigate the lithium–air battery, to monitor the evolution of the electrochemical products formed during cycling, and to gain insight into processes affecting capacity fading. While lithium peroxide is identified by 17O solid state NMR (ssNMR) as the predominant product in the first discharge in 1,2-dimethoxyethane (DME) based electrolytes, it reacts with the carbon cathode surface to form carbonate during the charging process. 13C ssNMR provides evidence for carbonate formation on the surface of the carbon cathode, the carbonate being removed at high charging voltages in the first cycle, but accumulating in later cycles. Small amounts of lithium hydroxide and formate are also detected in discharged cathodes and while the hydroxide formation is reversible, the formate persists and accumulates in the cathode upon further cycling. The results indicate that the rechargeability of the battery is limited by both the electrolyte and the carbon cathode stability. The utility of ssNMR spectroscopy in directly detecting product formation and decomposition within the battery is demonstrated, a necessary step in the assessment of new electrolytes, catalysts, and cathode materials for the development of a viable lithium–oxygen battery.

Recent advances in solid-state nuclear magnetic resonance techniques to quantify biomolecular dynamics.

The emergence of solid-state NMR (SSNMR) techniques over the last decade has greatly expanded the range of accessible biomolecules amenable to structural study.1-2 These methods were developed and first applied to a variety of microcrystalline model proteins including BPTI,3 SH3,4-5 ubiquitin,6-7 kaliotoxin,8 and GB1.9-11 These studies demonstrated that individual 13C, 15N and 1H sites throughout the entire protein could be uniquely resolved and assigned for purposes of high-resolution structure determination. Whereas traditional solid-state NMR approaches required site-specific isotopic labeling, the new developments enable entire proteins to be examined by resolving signals in multiple dimensions. These capabilities also permit the measurement of dynamic parameters through correlation spectroscopy, which increases the throughput, sensitivity, reliability and reproducibility of such measurements. A major advantage of SSNMR is that a large range of sample conditions and types can be examined, including many that are not accessible to solution NMR methods, such as membrane proteins and fibrils in addition to microcrystals. Recent examples include fibrils of the HET-s prion,12 high molecular weight oligomers of αB crystallin,13 the tetrameric assembly of the M2 peptide with bound amantadine drug in physiologically relevant bilayers,14 the trimeric membrane protein YadA,15 the photoreceptor proteorhodopsin,16 fibrils of the core domain of transthyretin,17 and beta-amyloid plaques derived from the brains of Alzheimer's disease patients.18 Such studies have opened up new avenues for exploration of fundamental biophysics and biochemistry, and yielded insights into clinically relevant events in membranes and aggregated states not accessible to solution NMR or X-ray crystallography. Moreover, SSNMR experiments are applicable over a larger range of temperatures than can be accessed by solution NMR19 and thereby enable a continuum of experimental conditions from physiological temperatures to the cryogenic temperatures at which most crystal structures are solved. Another advantage of NMR methods in general is that they are non-perturbing and do not require covalent modification of the protein. Thus, the dynamic information procured from such experiments has the potential to accurately represent the native protein behavior. This review focuses on developments over the last five years, regarding methods and applications of dynamics studies by solid-state NMR over timescales ranging from nanoseconds to days.

Experiments Optimized for Magic Angle Spinning and Oriented Sample Solid-State NMR of Proteins

Structure determination by solid-state NMR of proteins is rapidly advancing as a result of recent developments of samples, experimental methods, and calculations. There are a number of different solid-state NMR approaches that utilize stationary samples, aligned samples, or magic angle spinning of unoriented "powder" samples, and depending on the sample and the experimental method they can emphasize the measurement of distances or angles, ideally both, as sources of structural constraints. Multidimensional correlation spectroscopy of low-gamma nuclei such as (15)N and (13)C is an important step for making resonance assignments and measurements of angular restraints in membrane proteins. However, the efficiency of coherence transfer predominantly depends upon the strength of the dipole-dipole interaction, and this can vary from site to site and between sample alignments, for example, during the mixing of (13)C and (15)N magnetization in stationary aligned and in magic angle spinning samples. Here, we demonstrate that the efficiency of polarization transfer can be improved by using adiabatic demagnetization and remagnetization techniques on stationary aligned samples, and proton assisted insensitive nuclei cross-polarization in magic angle sample spinning samples. The adiabatic cross-polarization technique provides an alternative mechanism for spin-diffusion experiments correlating (15)N/(15)N and (15)N/(13)C chemical shifts over large distances. Improved efficiency in cross-polarization with 40-100% sensitivity enhancements is observed in proteins and single crystals, respectively. We describe solid-state NMR experimental techniques that are optimal for membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. The techniques are illustrated with data from single crystals both of peptides and of membrane proteins in phospholipid bilayers.

Solution and Solid State NMR Approaches To Draw Iron Pathways in the Ferritin Nanocage

Ferritins are intracellular proteins that can store thousands of iron(III) ions as a solid mineral. These structures autoassemble from four-helix bundle subunits to form a hollow sphere and are a prototypical example of protein nanocages. The protein acts as a reservoir, encapsulating iron as ferric oxide in its central cavity in a nontoxic and bioavailable form. Scientists have long known the structural details of the protein shell, owing to very high resolution X-ray structures of the apoform. However, the atomic level mechanism governing the multistep biomineralization process remained largely elusive. Through analysis of the chemical behavior of ferritin mutants, chemists have found the role of some residues in key reaction steps. Using Mössbauer and XAS, they have identified some di-iron intermediates of the catalytic reaction trapped by rapid freeze quench. However, structural information about the iron interaction sites remains scarce. The entire process is governed by a number of specific, but weak, interactions between the protein shell and the iron species moving across the cage. While this situation may constitute a major problem for crystallography, NMR spectroscopy represents an optimal tool to detect and characterize transient species involving soluble proteins. Regardless, NMR analysis of the 480 kDa ferritin represents a real challenge. Our interest in ferritin chemistry inspired us to use an original combination of solution and solid state approaches. While the highly symmetric structure of the homo-24-mer frog ferritin greatly simplifies the spectra, the large protein size hinders the efficient coherence transfer in solution, thus preventing the sequence specific assignments. In contrast, extensive (13)C-spin diffusion makes the solution (13)C-(13)C NOESY experiment our gold standard to monitor protein side chains both in the apoprotein alone and in its interaction with paramagnetic iron species, inducing line broadening on the resonances of nearby residues. We could retrieve the structural information embedded in the (13)C-(13)C NOESY due to a partial sequence specific assignment of protein backbone and side chains we obtained from solid state MAS NMR of ferritin microcrystals. We used the 59 assigned amino acids (∼33% of the total) as probes to locate paramagnetic ferric species in the protein cage. Through this approach, we could identify ferric dimers at the ferroxidase site and on their pathway towards the nanocage. Comparison with existing data on bacterioferritins and bacterial ferritins, as well as with eukaryotic ferritins loaded with various nonfunctional divalent ions, allowed us to reinterpret the available information. The resulting picture of the ferroxidase site is slightly different with various ferritins but is designed to provide multiple and generally weak iron ligands. The latter assist binding of two incoming iron(II) ions in two proximal positions to facilitate coupling with oxygen. Subsequent oxidation is accompanied by a decrease in the metal-metal distance (consistent with XAS/Mössbauer) and in the number of protein residues involved in metal coordination, facilitating the release of products as di-iron clusters under the effect of new incoming iron(II) ions.