Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus.

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure.

A General Concept for the Electronic and Steric Modification of 1-Metallacyclobuta-2,3-dienes: A Case Study of Group 4 Metallocene Complexes.

The synthesis of group 4 metal 1-metallacyclobuta-2,3-dienes as organometallic analogues of elusive 1,2-cyclobutadiene has so far been limited to SiMe3 substituted examples. We present the synthesis of two Ph substituted dilithiated ligand precursors for the preparation of four new 1-metallacyclobuta-2,3-dienes [rac-(ebthi)M] (M=Ti, Zr; ebthi=1,2-ethylene-1,10-bis(η5-tetrahydroindenyl)). The organolithium compounds [Li2(RC3Ph)] (1 b: R=Ph, 1 c: R=SiMe3) as well as the metallacycles of the general formula [rac-(ebthi)M(R1C3R2)] (2 b: M=Ti, R1=R2=Ph, 2 c: M=Ti, R1=Ph, R2=SiMe3; 3 b: M=Zr, R1=R2=Ph; 3 c: M=Zr, R1=Ph, R2=SiMe3) were fully characterised. Single crystal X-ray diffraction and quantum chemical bond analysis of the Ti and Zr complexes reveal ligand influence on the biradicaloid character of the titanocene complexes. X-band EPR spectroscopy of structurally similar Ti complexes [rac-(ebthi)Ti(Me3SiC3SiMe3)] (2 a), 2 b, and 2 c was carried out to evaluate the accessibility of an EPR active triplet state. Cyclic voltammetry shows that introduction of Ph groups renders the complexes easier to reduce. 13C CPMAS NMR analysis provides insights into the cause of the low field shift of the resonances of metal-bonded carbon atoms and provides evidence of the absence of the ß-C-Ti interaction.

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.

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics.

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA's secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.

Rational Design of a Phosphorus-Centered Disbiradical.

Phosphorus-centered disbiradicals, in which the radical sites exist as individual spin doublets with weak spin-spin interaction have not been known so far. Starting from monoradicals of the type [⋅P(µ-NTer)2 P-R], we have now succeeded in linking two such monoradical phosphorus centers by appropriate choice of a linker. To this end, biradical [⋅P(µ-NTer)2 P⋅] (1) was treated with 1,6-dibromohexane, affording the brominated species {Br[P(µ-NTer)]2 }2 C6 H12 (3). Subsequent reduction with KC8 led to the formation of the disbiradical {⋅[P(µ-NTer)]2 }2 C6 H12 (4) featuring a large distance between the radical phosphorus sites in the solid state and formally the highest biradical character observed in a P-centered biradical so far, approaching 100 %. EPR spectroscopy revealed a three-line signal in solution with a considerably larger exchange interaction than would be expected from the molecular structure of the single crystal. Quantum chemical calculations revealed a highly dynamic conformational space; thus, the two radical sites can approach each other with a much smaller distance in solution. Further reduction of 4 resulted in the formation of a potassium salt featuring the first structurally characterized P-centered distonic radical anion (5- ). Moreover, 4 could be used in small molecule activation.

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.

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.

Serial Polarization Transfer by Combination of Cross-Relaxation and Rotational Resonance for Sensitivity-Enhanced Solid-State NMR.

Methods which induce site-specificity and sensitivity enhancement in solid-state magic-angle spinning NMR spectroscopy become more important for structural biology due to the increasing size of molecules under investigation. Recently, several strategies have been developed to increase site specificity and thus reduce signal overlap. Under dynamic nuclear polarization (DNP) for NMR signal enhancement, it is possible to use cross-relaxation transfer induced by select dynamic groups within the molecules which is exploited by SCREAM-DNP (Specific Cross Relaxation Enhancement by Active Motions under DNP). Here, we present an approach where we additionally reintroduce the homonuclear dipolar coupling with rotational resonance (R2 ) during SCREAM-DNP to further boost the selectivity of the experiment. Detailed analysis of the polarization buildup dynamics of 13 C-methyl polarization source and 13 C-carbonyl target in 2-13 C-ethyl 1-13 C-acetate provides information about the sought-after and spurious transfer pathways. We show that dipolar-recoupled transfer rates greatly exceed the DNP buildup dynamics in our model system, indicating that significantly larger distances can be selectively and efficiently hyperpolarized.

Serial Polarization Transfer by Combination of Cross-Relaxation and Rotational Resonance for Sensitivity-Enhanced Solid-State NMR.

The front cover artwork is provided by Prof. Björn Corzilius's group at the University of Rostock. The image shows the interference of the isotropic frequency difference between two nuclear spins with the evolution of their dipolar coupling in an MAS rotor. This rotational resonance (R2 ) can be exploited for site-specific dynamic nuclear polarization. Read the full text of the Research Article at 10.1002/cphc.202300206.

Electron-decoupled MAS DNP with N@C60.

Frequency-chirped microwaves decouple electron- and 13C-spins in magic-angle spinning N@C60:C60 powder, improving DNP-enhanced 13C NMR signal intensity by 12% for 7 s polarization, and 5% for 30 s polarization. This electron decoupling demonstration is a step toward utilizing N@C60 as a controllable electron-spin source for magic-angle spinning magnetic resonance experiments.

High-Field Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) is one of the most prominent methods of sensitivity enhancement in nuclear magnetic resonance (NMR). Even though solid-state DNP under magic-angle spinning (MAS) has left the proof-of-concept phase and has become an important tool for structural investigations of biomolecules as well as materials, it is still far from mainstream applicability because of the potentially overwhelming combination of unique instrumentation, complex sample preparation, and a multitude of different mechanisms and methods available. In this review, I introduce the diverse field and history of DNP, combining aspects of NMR and electron paramagnetic resonance. I then explain the general concepts and detailed mechanisms relevant at high magnetic field, including solution-state methods based on Overhauser DNP but with a greater focus on the more established MAS DNP methods. Finally, I review practical considerations and fields of application and discuss future developments.

The influence of the cation structure on the basicity-related polarity of ionic liquids.

UV/Vis absorption data of (E)-4-(2-[5-{4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl}thiene-2-yl]vinyl)-2-(dicyano-methylene)-3-cyano-5,5-dimethyl-2,5-dihydrofuran (ThTCF) as a solvatochromic probe is applied to examine the anion coordination strength (e.g. of N(CN)2, BF4, PF6, N(Tf)2, CF3COO) as a function of the cation structure of ionic liquids. Several 1-n-alky-3-methylimidazolium- and tetraalkylammonium CH3-NR3+-based ILs with different n-alkyl chain lengths (R = -C4H9, -C6H11, -C8H17, -C10H21) are considered. UV/Vis absorption data of ThTCF show subtle correlations with hydrogen bond accepting (HBA) ability-related measurands such as Kamlet-Taft ß, Freire's EHB, and Laurence ß1 parameter as a function of anion and cation structure. The different influence of the n-alkyl chain length of imidazolium- and tetraalkylammonium-based ILs on the dipolarity and HBA strength is confirmed by comparison with the 14N isotropic hyperfine coupling constants (Aiso) of a positively (CATI) and negatively charged spin probe (TSKCr) of TEMPO-type [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl] and quantum chemically derived dipoles of the cations. The Aiso values correlate with the absorption energy of ThTCF and EHB, but in different ways depending on the anion or charge of the spin probe. In a final discussion of the ß, EHB, and ß1 scales in relation to ThTCF, the importance of the molar concentration N of ionic liquids for the physical significance of the respective parameters is discussed.

Site-specific dynamic nuclear polarization in a Gd(III)-labeled protein.

Dynamic nuclear polarization (DNP) of a biomolecule tagged with a polarizing agent has the potential to not only increase NMR sensitivity but also to provide specificity towards the tagging site. Although the general concept has been often discussed, the observation of true site-specific DNP and its dependence on the electron-nuclear distance has been elusive. Here, we demonstrate site-specific DNP in a uniformly isotope-labeled ubiquitin. By recombinant expression of three different ubiquitin point mutants (F4C, A28C, and G75C) post-translationally modified with a Gd3+-chelator tag, localized metal-ion DNP of 13C and 15N is investigated. Effects counteracting the site-specificity of DNP such as nuclear spin-lattice relaxation and proton-driven spin diffusion have been attenuated by perdeuteration of the protein. Particularly for 15N, large DNP enhancement factors on the order of 100 and above as well as localized effects within side-chain resonances differently distributed over the protein are observed. By analyzing the experimental DNP built-up dynamics combined with structural modeling of Gd3+-tags in ubiquitin supported by paramagnetic relaxation enhancement (PRE) in solution, we provide, for the first time, quantitative information on the distance dependence of the initial DNP transfer. We show that the direct 15N DNP transfer rate indeed linearly depends on the square of the hyperfine interaction between the electron and the nucleus following Fermi's golden rule, however, below a certain distance cutoff paramagnetic signal bleaching may dramatically skew the correlation.

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.

Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor.

While uniform isotope labeling of ribonucleic acids (RNA) can simply and efficiently be achieved by in-vitro transcription, the specific introduction of nucleotides in larger constructs is non-trivial and often ineffective. Here, we demonstrate how a medium-sized (67-mer), biocatalytically relevant RNA (hammerhead ribozyme, HHRz) can be formed by spontaneous hybridization of two differently isotope-labeled strands, each individually synthesized by in-vitro transcription. This allows on the one hand for a significant reduction in the number of isotope-labeled nucleotides and thus spectral overlap particularly under magic-angle spinning (MAS) dynamic nuclear polarization (DNP) NMR conditions, on the other hand for orthogonal 13C/15N-labeling of complementary strands and thus for specific investigation of structurally or functionally relevant inter-strand and/or inter-stem contacts. By this method, we are able to confirm a non-canonical interaction due to single-site resolution and unique spectral assignments by two-dimensional 13C-13C (PDSD) as well as 15N-13C (TEDOR) correlation spectroscopy under "conventional" DNP enhancement. This contact is indicative of the ribozyme's functional conformation, and is present in frozen solution irrespective of the presence or absence of a Mg2+ co-factor. Finally, we use different isotope-labeling schemes in order to investigate the distance dependence of paramagnetic interactions and direct metal-ion DNP if the diamagnetic Mg2+ is substituted by paramagnetic Mn2+.

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.

Probing Interactions of N-Donor Molecules with Open Metal Sites within Paramagnetic Cr-MIL-101: A Solid-State NMR Spectroscopic and Density Functional Theory Study.

Understanding host-guest interactions is one of the key requirements for adjusting properties in metal-organic frameworks (MOFs). In particular, systems with coordinatively unsaturated Lewis acidic metal sites feature highly selective adsorption processes. This is attributed to strong interactions with Lewis basic guest molecules. Here we show that a combination of 13C MAS NMR spectroscopy with state-of-the-art density functional theory (DFT) calculations allows one to unravel the interactions of water, 2-aminopyridine, 3-aminopyridine, and diethylamine with the open metal sites in Cr-MIL-101. The 13C MAS NMR spectra, obtained with ultrafast magic-angle spinning, are well resolved, with resonances distributed over 1000 ppm. They present a clear signature for each guest at the open metal sites. Based on competition experiments this leads to the following binding preference: water < diethylamine ≈ 2-aminopyridine < 3-aminopyridine. Assignments were done by exploiting distance sum relations derived from spin-lattice relaxation data and 13C{1H} REDOR spectral editing. The experimental data were used to validate NMR shifts computed for the Cr-MIL-101 derivatives, which contain Cr3O clusters with magnetically coupled metal centers. While both approaches provide an unequivocal assignment and the arrangement of the guests at the open metal sites, the NMR data offer additional information about the guest and framework dynamics. We expect that our strategy has the potential for probing the binding situation of adsorbate mixtures at the open metal sites of MOFs in general and thus accesses the microscopic interaction mechanisms for this important material class, which is essential for deriving structure-property relationships.

Efficiency of Water-Soluble Nitroxide Biradicals for Dynamic Nuclear Polarization in Rotating Solids at 9.4†T: bcTol-M and cyolyl-TOTAPOL as New Polarizing Agents.

Nitroxide biradicals are very efficient polarizing agents in magic angle spinning (MAS) cross effect (CE) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR). Many recently synthesized, new radicals show superior DNP-efficiency in organic solvents but suffer from insufficient solubility in water or glycerol/water for biological applications. We report DNP efficiencies for two new radicals, the water-soluble bcTol-M and cyolyl-TOTAPOL, and include a comparison with three known biradicals, TOTAPOL, bcTol, and AMUPol. They differ by linker groups, featuring either a 3-aminopropane-1,2-diol or a urea tether, or by the structure of the alkyl substituents that flank the nitroxide groups. For evaluating their performances, we measured both signal enhancements ϵ and DNP-enhanced sensitivity κ, and compared the results to electron spin relaxation data recorded at the same magnetic field strength (9.4 T). In our study, differences in DNP efficiency correlate with changes in the nuclear polarization dynamics rather than electron relaxation. The ratios of their individual ϵ and κ differ by up to 20 %, which is explained by starkly different nuclear polarization build-up rates. For the radicals compared here empirically, using proline standard solutions, the new radical bcTol-M performs best while being most soluble in water/glycerol mixtures.

Electron-driven spin diffusion supports crossing the diffusion barrier in MAS DNP.

Dynamic nuclear polarization (DNP) can be applied to enhance the sensitivity of solid-state NMR experiments by several orders of magnitude due to microwave-driven transfer of spin polarization from unpaired electrons to nuclei. While the underlying quantum mechanical aspects are sufficiently well understood on a microscopic level, the exact description of the large-scale spin dynamics, usually involving hundreds to thousands of nuclear spins per electron, is still lacking consensus. Generally, it is assumed that nuclear hyperpolarization can only be observed on nuclei which do not experience strong influence of the unpaired electrons and thus being significantly removed from the paramagnetic polarizing agents. At the same time, sufficiently strong hyperfine interaction is required for DNP transfer. Therefore, efficient nuclear spin diffusion from the strongly-interacting nuclei to the NMR-observable bulk is considered to be essential for efficient nuclear hyperpolarization. Based on experimental results obtained on the endohedral fullerene N@C60 as a polarizing agent sparsely diluted in C60, we discuss the effect of the spin-diffusion barrier. We introduce electron-driven spin diffusion (EDSD) as a novel mechanism for nuclear polarization transfer in the proximity of an electron spin which is particularly relevant under magic-angle spinning (MAS) DNP conditions.