Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR.

Solid-state NMR with magic-angle spinning (MAS) is an important method in structural biology. While NMR can provide invaluable information about local geometry on an atomic scale even for large biomolecular assemblies lacking long-range order, it is often limited by low sensitivity due to small nuclear spin polarization in thermal equilibrium. Dynamic nuclear polarization (DNP) has evolved during the last decades to become a powerful method capable of increasing this sensitivity by two to three orders of magnitude, thereby reducing the valuable experimental time from weeks or months to just hours or days; in many cases, this allows experiments that would be otherwise completely unfeasible. In this review, we give an overview of the developments that have opened the field for DNP-enhanced biomolecular solid-state NMR including state-of-the-art applications at fast MAS and high magnetic field. We present DNP mechanisms, polarizing agents, and sample constitution methods suitable for biomolecules. A wide field of biomolecular NMR applications is covered including membrane proteins, amyloid fibrils, large biomolecular assemblies, and biomaterials. Finally, we present perspectives and recent developments that may shape the field of biomolecular DNP in the future.

Specific Signal Enhancement on an RNA-Protein Interface by Dynamic Nuclear Polarization.

Invited for the cover of this issue are the groups of Alexander Marchanka at the Leibniz University of Hannover and Björn Corzilius at the University of Rostock. The image depicts the local generation of nuclear spin hyperpolarization, which selectively "illuminates" the interaction surface of a ribonuclear protein complex for solid-state NMR spectroscopy. Read the full text of the article at 10.1002/chem.202203443.

Specific Signal Enhancement on an RNA-Protein Interface by Dynamic Nuclear Polarization.

Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13 C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP.

Exploring Protein Structures by DNP-Enhanced Methyl Solid-State NMR Spectroscopy.

Although the rapid development of sensitivity-enhanced solid-state NMR (ssNMR) spectroscopy based on dynamic nuclear polarization (DNP) has enabled a broad range of novel applications in material and life sciences, further methodological improvements are needed to unleash the full potential of DNP-ssNMR. Here, a new methyl-based toolkit for exploring protein structures is presented, which combines signal-enhancement by DNP with heteronuclear Overhauser effect (hetNOE), carbon-carbon-spin diffusion (SD) and strategically designed isotope-labeling schemes. It is demonstrated that within this framework, methyl groups can serve as dynamic sensors for probing local molecular packing within proteins. Furthermore, they can be used as "NMR torches" to selectively enlighten their molecular environment, e.g., to selectively enhance the polarization of nuclei within residues of ligand-binding pockets. Finally, the use of 13C-13C spin diffusion enables probing carbon-carbon distances within the subnanometer range, which bridges the gap between conventional 13C-ssNMR methods and EPR spectroscopy. The applicability of these methods is directly shown on a large membrane protein, the light-driven proton pump green proteorhodopsin (GPR), which offers new insight into the functional mechanism of the early step of its photocycle.

Methyl dynamics in amino acids modulate heteronuclear cross relaxation in the solid state under MAS DNP.

Dynamic Nuclear Polarization (DNP) is a wide-spread technique for sensitivity enhancement of MAS NMR. During a typical MAS DNP experiment, several mechanisms resulting in polarization transfer may be active at the same time. One such mechanism which is most commonly active but up to now mostly disregarded is SCREAM-DNP (Specific Cross Relaxation Enhancement by Active Motions under DNP). This effect is generally observed in direct DNP experiments if molecular dynamics are supporting heteronuclear cross relaxation similar to the nuclear Overhauser effect. We investigate this effect for the CH3 groups of all methyl-bearing amino acids (i.e., alanine, valine, leucine, isoleucine, threonine, and methionine). At the typical DNP temperature of ∼110 K the three-fold reorientation dynamics are still active, and efficient SCREAM-DNP is observed. We discuss variations in enhancement factors obtained by this effect in context of sample temperature and sterical hindrance of the methyl group. Next to the direct transfer to the methyl carbon, we also find evidence for much weaker transfer from the methyl protons directly to other carbons in the amino acid molecule and succeed to correlate build-up dynamics with the CH dipole coupling which is modulated by the CH3 orientation. Besides methyl dynamics we also identify ring dynamics within proline as a source of SCREAM-DNP. Our results are the first step towards utilization of this effect as a specific probing techniqueusing methyl groups in protein systems.

Complex Formation of the Tetracycline-Binding Aptamer Investigated by Specific Cross-Relaxation under DNP.

While dynamic nuclear polarization (DNP) under magic-angle spinning (MAS) is generally a powerful method capable of greatly enhancing the sensitivity of solid-state NMR spectroscopy, hyperpolarization also gives rise to peculiar spin dynamics. Here, we elucidate how specific cross-relaxation enhancement by active motions under DNP (SCREAM-DNP) can be utilized to selectively obtain MAS-NMR spectra of an RNA aptamer in a tightly bound complex with a methyl-bearing ligand (tetracycline) due to the effective CH3 -reorientation at an optimized sample temperature of approximately 160 K. SCREAM-DNP can spectrally isolate the complex from non-bound species in an RNA mixture. This selectivity allows for a competition assay between the aptamer and a mutant with compromised binding affinity. Variations in molecular structure and methyl dynamics, as observed by SCREAM-DNP, between free tetracycline and RNA-bound tetracycline are discussed.

Heteronuclear Cross-Relaxation under Solid-State Dynamic Nuclear Polarization.

We report on the spontaneous polarization transfer from dynamically hyperpolarized 1H to 13C during magic-angle spinning dynamic nuclear polarization (DNP) at temperatures around 100 K. The transfer is mediated by 1H-13C cross-relaxation within methyl groups due to reorientation dynamics, and results in an inverted 13C NMR signal of enhanced amplitude. Further spreading of transferred polarization can then occur via 13C-13C spin-diffusion. The resulting process is equal to the nuclear Overhauser effect (NOE) where typically continuous saturation of 1H by radio frequency irradiation is employed. Here, hyperpolarization by irradiation with microwaves in the presence of typical bis-nitroxide polarizing agents is utilized for steady-state displacement of 1H polarization from thermal equilibrium and perpetual spin-lattice relaxation. An effective 13C enhancement factor of up to -15 has been measured. Presence of Gd(III) furthermore amplifies the effect likely by accelerated relaxation of 1H. We provide experimental evidence for the proposed mechanism and show that DNP-induced cross-relaxation is a robust feature within proteins and single amino acids and discuss potential applications.

Molecular differences in collagen organization and in organic-inorganic interfacial structure of bones with and without osteocytes.

Bone is a fascinating biomaterial composed mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, and water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications for the mechanical properties of the particular bone. STATEMENT OF SIGNIFICANCE: Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.

Coagulation using organic carbonates opens up a sustainable route towards regenerated cellulose films.

Due to their biodegradability, biocompatibility and sustainable nature, regenerated cellulose (RC) films are of enormous relevance for green applications including medicinal, environmental and separation technologies. However, the processes used so far are very hazardous to the environment and health. Here, we disclose a simple, fast, environmentally friendly, nontoxic and cost-effective processing method for preparing RC films. High quality non-transparent and transparent RC films and powders can be produced by dissolution with tetrabutylphosphonium hydroxide [TBPH]/[TBP]+[OH]- followed by coagulation with organic carbonates. Investigations on the coagulation mechanism revealed an extremely fast reaction between the carbonates and the hydroxide ions. The high-quality powders and films were fully characterized with respect to structure, surface morphology, permeation and selectivity. This method represents a future-oriented green alternative to known industrial processes.