Blue emissive amidinate-based tetra-coordinated boron compounds.

A series of novel amidinate ligated four-coordinated boron compounds, [(Ar)-C(tBuN)2BF2] (1BF2-6BF2), were synthesised and structurally characterised (Ar = 1-phenyl, 2-naphthyl, 2-anthryl, 9-anthryl, 9-phenanthryl and 1-pyrene). The increased π-conjugation of Ar-substitution on the amidinate ligand results in dark blue-emission in compounds 3BF2-6BF2. All these compounds are emissive in the solution state. The 2-anthryl substituted compound 3BF2 was found to exhibit a maximum quantum yield of 48% in dichloromethane. Theoretical studies were carried out which validate the hypothesis about the increased π-conjugation.

Reduction of 2-H-substituted pyrrolinium cations: the carbon-carbon single bond in air stable 2,2'-bipyrrolidines as a two-electron-source.

Reduction of 2-H-substituted pyrrolinium cations via initially formed secondary radicals results in either dimerisation or H-abstracted products, while the outcome depends on the N-substituents. The resultant central carbon-carbon single bond in the dimerised 2,2'-bipyrrolidine derivatives can be oxidised chemically and electrochemically. The notably air and moisture-stable dimers were subsequently utilised as a source of two electrons in various chemical transformations.

Measuring Dipolar Order Parameters in Nondeuterated Proteins Using Solid-State NMR at the Magic-Angle-Spinning Frequency of 100 kHz.

Proteins are dynamic molecules, relying on conformational changes to carry out function. Measurement of these conformational changes can provide insight into how function is achieved. For proteins in the solid state, this can be done by measuring the decrease in the strength of anisotropic interactions due to motion-induced fluctuations. The measurement of one-bond heteronuclear dipole-dipole coupling at magic-angle-spinning (MAS) frequencies >60 kHz is ideal for this purpose. However, rotational-echo double resonance (REDOR), an otherwise gold-standard technique for the quantitative measurement of these couplings, is difficult to implement under these conditions, especially in nondeuterated samples. We present here a combination of strategies based on REDOR variants ϵ-REDOR and DEDOR (deferred REDOR) and simultaneously measure residue-specific 15N-1H and 13Cα-1Hα dipole-dipole couplings in nondeuterated systems at the MAS frequency of 100 kHz. These strategies open up avenues to access dipolar order parameters in a variety of systems at the increasingly fast MAS frequencies that are now available.

Asynchronising five-fold symmetry sequence for better homonuclear polarisation transfer in magic-angle-spinning solid-state NMR.

Recoupling, decoupling, and multidimensional correlation experiments in magic-angle-spinning (MAS) solid-state NMR can be designed by exploiting the symmetry of internal spin interactions. One such scheme, namely, C521, and its supercycled version SPC521, notated as a five-fold symmetry sequence, is widely used for double-quantum dipole-dipole recoupling. Such schemes are generally rotor synchronised by design. We demonstrate an asynchronous implementation of the SPC521 sequence leading to higher double-quantum homonuclear polarisation transfer efficiency compared to the normal synchronous implementation. Rotor-synchronisation is broken in two different ways: lengthening the duration of one of the pulses, denoted as pulse-width variation (PWV), and mismatching the MAS frequency denoted as MAS variation (MASV). The application of this asynchronous sequence is shown on three different samples, namely, U-13C-alanine and 1,4-13C-labelled ammonium phthalate which include 13Cα-13Cß, 13Cα-13Co, and 13Co-13Co spin systems, and adenosine 5'- triphosphate disodium salt trihydrate (ATP⋅3H2O). We show that the asynchronous version performs better for spin pairs with small dipole-dipole couplings and large chemical-shift anisotropies, for example, 13Co-13Co. Simulations and experiments are shown to corroborate the results.

Assignment of aromatic side-chain spins and characterization of their distance restraints at fast MAS.

The assignment of aromatic side-chain spins has always been more challenging than assigning backbone and aliphatic spins. Selective labeling combined with mutagenesis has been the approach for assigning aromatic spins. This manuscript reports a method for assigning aromatic spins in a fully protonated protein by connecting them to the backbone atoms using a low-power TOBSY sequence. The pulse sequence employs residual polarization and sequential acquisitions techniques to record HN- and HC-detected spectra in a single experiment. The unambiguous assignment of aromatic spins also enables the characterization of 1H-1H distance restraints involving aromatic spins. Broadband (RFDR) and selective (BASS-SD) recoupling sequences were used to generate HN-ΗC, HC-HN and HC-HC restraints involving the side-chain proton spins of aromatic residues. This approach has been demonstrated on a fully protonated U-[13C,15N] labeled GB1 sample at 95-100 kHz MAS.

Mechanism of selective polarization exchange amongst chemically similar and distinct protons during weak rf irradiation at fast magic angle spinning.

Band Selective Spectral Spin-Diffusion (BASS-SD) is a method to obtain selective 1H-1H contacts between chemically similar protons within a distance range of 5-6 Å in fully protonated proteins. BASS-SD combines low-amplitude proton spinlock radio frequency (rf) pulses with fast MAS frequency to enable selective polarization exchange in fully protonated molecules. The selectivity of transfer is dictated by the bandwidth of the spinlock pulse and has been used to observe selective HN-HN, Hα-Ηα and Hmethyl-Hmethyl correlations. These proton-proton spatial contacts are similar to those observed in perdeuterated samples and serve as useful structural restraints towards de novo protein structure determination. This study employs bimodal Floquet theory to derive the first- and second-order effective Hamiltonians necessary to understand the spin dynamics during BASS-SD. Analytical calculations combined with numerical simulations delineate two different mechanisms for polarization transfer amongst the proton spins. The BASS-SD recoupling condition has been reoptimized to observe selective correlations between chemically different protons (e.g., HN-Hα) while retaining the spatial contacts between chemically similar protons (e.g., HN-HN). The new BASS-SD condition is integrated with simultaneous and sequential acquisition approaches to generate four different types of structural restraints (HN-HN, Hα-Ηα, HN-Hα, Hα-HN) in one experiment. The approach has been demonstrated on microcrystalline U-[13C,15N] labeled GB1 protein at âˆ¼ 95-100 kHz MAS.

CURD: a Single-Shot Strategy to Obtain Assignments and Distance Restraints for Proteins Using Solid-State MAS NMR Spectroscopy.

We present a strategy dubbed CURD (correlations using recycle delays) to acquire chemical-shift assignments and distance restraints for proteins in a single experimental block under slow-moderate magic-angle spinning conditions. This is done by concatenating the 3D-CCC and 3D-NNC experiments, both of which individually require long experimental times for sufficient resolution and sensitivity to be realized. Unlike previous approaches, the CURD strategy does not increase the amount of radio-frequency deposition on the sample and does not require lengthy procedures to optimize any of the pulse sequence elements. Instead, time savings is obtained by using the hitherto unused recycle delay of one of the experiments (2D-CC/3D-CCC) to establish inter-residue correlations for the second experiment (2D-NN/3D-NNC). Experiments are demonstrated on a model protein at the MAS frequency of 12.5 kHz and are shown to result in time savings of the order of days for most of the routine cases.

Solid-State NMR: Methods for Biological Solids.

In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100's of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

A comparison between MBP- and NT* as N-terminal fusion partner for recombinant protein production in E. coli.

Advances in structural biology have been fueled in part by developing techniques for large-scale heterologous expression and purification of proteins. Nevertheless, this step is still a bottleneck in biophysical studies of many proteins. Often, fusion proteins are used to increase expression levels, solubility, or both. Here, we compare a recently reported fusion tag, NT*, with Maltose Binding Protein (MBP), a well-known fusion tag and solubility enhancer. NT* shows high expression and solubility when used as an N-terminal fusion partner for several aggregation-prone peptides. Its efficacy in enhancing the solubility of aggregation-prone globular proteins has, however, not been tested. We find here that although the overall expression levels for NT* fusions are much higher than those for the MBP fusion, MBP was far superior for enhancing the solubility of the passenger protein. Nevertheless, the effective yield after purification from the soluble fraction of both MBP-fusion and NT*-fusion was comparable, mainly due to higher expression levels in NT*-fusion and a smaller fraction of the passenger protein net weight being locked in the fusion protein. We conclude that NT* is an excellent fusion tag to improve the overall expression of globular proteins but does not increase the passenger protein's solubility compared to MBP. Proteins that are partially soluble or can be refolded in-vitro will significantly benefit from N-terminal NT* fusions. MBP, however, still remains one of the very few options for an N-terminal fusion if the solubility of the protein after expression is critical for preserving its proper fold or activity.

Probing the Influence of Single-Site Mutations in the Central Cross-ß Region of Amyloid ß (1-40) Peptides.

Amyloid ß (Aß) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer's disease. A complete mechanistic understanding how Aß peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aß40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aß assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aß40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aß fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aß fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aß40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aß40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aß fibrillation pathway.

Floquet theory in magnetic resonance: Formalism and applications.

Floquet theory is an elegant mathematical formalism originally developed to solve time-dependent differential equations. Besides other fields, it has found applications in optical spectroscopy and nuclear magnetic resonance (NMR). This review attempts to give a perspective of the Floquet formalism as applied in NMR and shows how it allows one to solve various problems with a focus on solid-state NMR. We include both matrix- and operator-based approaches. We discuss different problems where the Hamiltonian changes with time in a periodic way. Such situations occur, for example, in solid-state NMR experiments where the time dependence of the Hamiltonian originates either from magic-angle spinning or from the application of amplitude- or phase-modulated radiofrequency fields, or from both. Specific cases include multiple-quantum and multiple-frequency excitation schemes. In all these cases, Floquet analysis allows one to define an effective Hamiltonian and, moreover, to treat cases that cannot be described by the more popularly used and simpler-looking average Hamiltonian theory based on the Magnus expansion. An important example is given by spin dynamics originating from multiple-quantum phenomena (level crossings). We show that the Floquet formalism is a very general approach for solving diverse problems in spectroscopy.

Structural basis for sarcolipin's regulation of muscle thermogenesis by the sarcoplasmic reticulum Ca2+-ATPase.

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) plays a central role in muscle contractility and nonshivering thermogenesis. SERCA is regulated by sarcolipin (SLN), a single-pass membrane protein that uncouples Ca2+ transport from ATP hydrolysis, promoting futile enzymatic cycles and heat generation. The molecular determinants for regulating heat release by the SERCA/SLN complex are unclear. Using thermocalorimetry, chemical cross-linking, and solid-state NMR spectroscopy in oriented phospholipid bicelles, we show that SERCA's functional uncoupling and heat release rate are dictated by specific SERCA/SLN intramembrane interactions, with the carboxyl-terminal residues anchoring SLN to the SR membrane in an inhibitory topology. Systematic deletion of the carboxyl terminus does not prevent the SERCA/SLN complex formation but reduces uncoupling in a graded manner. These studies emphasize the critical role of lipids in defining the active topology of SLN and modulating the heat release rate by the SERCA/SLN complex, with implications in fat metabolism and basal metabolic rate.

Multiplexing experiments in NMR and multi-nuclear MRI.

Multiplexing NMR experiments by direct detection of multiple free induction decays (FIDs) in a single experiment offers a dramatic increase in the spectral information content and often yields significant improvement in sensitivity per unit time. Experiments with multi-FID detection have been designed with both homonuclear and multinuclear acquisition, and the advent of multiple receivers on commercial spectrometers opens up new possibilities for recording spectra from different nuclear species in parallel. Here we provide an extensive overview of such techniques, designed for applications in liquid- and solid-state NMR as well as in hyperpolarized samples. A brief overview of multinuclear MRI is also provided, to stimulate cross fertilization of ideas between the two areas of research (NMR and MRI). It is shown how such techniques enable the design of experiments that allow structure elucidation of small molecules from a single measurement. Likewise, in biomolecular NMR experiments multi-FID detection allows complete resonance assignment in proteins. Probes with multiple RF microcoils routed to multiple NMR receivers provide an alternative way of increasing the throughput of modern NMR systems, effectively reducing the cost of NMR analysis and increasing the information content at the same time. Solid-state NMR experiments have also benefited immensely from both parallel and sequential multi-FID detection in a variety of multi-dimensional pulse schemes. We are confident that multi-FID detection will become an essential component of future NMR methodologies, effectively increasing the sensitivity and information content of NMR measurements.

Activation of Aromatic C-F Bonds by a N-Heterocyclic Olefin (NHO).

A N-heterocyclic olefin (NHO), a terminal alkene selectively activates aromatic C-F bonds without the need of any additional catalyst. As a result, a straightforward methodology was developed for the formation of different fluoroaryl-substituted alkenes in which the central carbon-carbon double bond is in a twisted geometry.

Overcoming Prohibitively Large Radiofrequency Demands for the Measurement of Internuclear Distances with Solid-State NMR under Fast Magic-Angle Spinning.

Solid-state NMR is a powerful tool to measure distances and motional order parameters which are vital tools in characterizing the structure and dynamics of molecules. Magic-angle spinning (MAS), widely employed in solid-state NMR, averages out dipole-dipole couplings that carry such information. Hence, rotor-synchronized radiofrequency (RF) pulses, that interfere with MAS averaging, are commonly employed to measure such couplings. However, most of the methods that achieve this, rotational echo double resonance (REDOR) being a classic example, require RF amplitudes that are greater than or equal to the MAS frequency. While feasible at MAS frequencies <40 kHz, these requirements become prohibitively large for higher MAS frequencies (40-110 kHz), which are now commercially available. Here, we redesign the REDOR experiment so that RF amplitudes as low as 0.5-0.7 times the spinning frequency can be used. This sequence, name deferred rotational echo double resonance (DEDOR), thus extends the utility of this method to the fastest MAS frequencies currently commercially available (111 kHz). The generality of this strategy is shown by extending it to other methods that utilize the same principle as REDOR. They will be useful in obtaining structural parameters for a wide range of molecules using solid-state NMR under fast MAS with the additional advantage of higher spectral resolution under these conditions.

Simultaneous recording of intra- and inter-residue linking experiments for backbone assignments in proteins at MAS frequencies higher than 60 kHz.

Obtaining site-specific assignments for the NMR spectra of proteins in the solid state is a significant bottleneck in deciphering their biophysics. This is primarily due to the time-intensive nature of the experiments. Additionally, the low resolution in the [Formula: see text]-dimension requires multiple complementary experiments to be recorded to lift degeneracies in assignments. We present here an approach, gleaned from the techniques used in multiple-acquisition experiments, which allows the recording of forward and backward residue-linking experiments in a single experimental block. Spectra from six additional pathways are also recovered from the same experimental block, without increasing the probe duty cycle. These experiments give intra- and inter residue connectivities for the backbone [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text] resonances and should alone be sufficient to assign these nuclei in proteins at MAS frequencies > 60 kHz. The validity of this approach is tested with experiments on a standard tripeptide N-formyl methionyl-leucine-phenylalanine (f-MLF) at a MAS frequency of 62.5 kHz, which is also used as a test-case for determining the sensitivity of each of the experiments. We expect this approach to have an immediate impact on the way assignments are obtained at MAS frequencies [Formula: see text].

On the direct relation between REDOR and DIPSHIFT experiments in solid-state NMR.

Rotational-echo double resonance (REDOR) and Dipolar-coupling chemical-shift correlation (DIPSHIFT) are commonly used experiments to probe heteronuclear dipole-dipole couplings between isolated pairs of spin-12 nuclei in magic-angle-spinning (MAS) solid-state NMR. Their widespread use is due to their robustness to experimental imperfections and a straightforward interpretation of data. Both of these experiments use rotor-synchronised π pulses to recouple the heteronuclear dipole-dipole couplings, and the observed intensity of resonances is modulated by a recoupled phase factor depending on the position or duration of the recoupling pulses. Several modifications to both of these experiments have been proposed, for example, the development of DIPSHIFT which employs strategies that mimic the multi-rotor-period nature of REDOR. We show here that REDOR and DIPSHIFT are in fact alternate implementations of the same experiment. The overt similarity in the design of REDOR and DIPSHIFT is also reflected in their theoretical description. Dipolar dephasing curves in REDOR are obtained by increasing the recoupling duration whilst keeping the position of the pulses constant, which results in a dephasing factor that is a function of only the dephasing time. DIPSHIFT, on the other hand, is a constant-time version of REDOR; the dipolar dephasing is a function of the position of the pulses with respect to the rotor period. We discuss the advantages and disadvantages of each implementation and suggest domains of applicability for these sequences.

Experiments with direct detection of multiple FIDs.

Pulse schemes with direct observation of multiple free induction decays (FIDs) offer a dramatic increase in the spectral information content of NMR experiments and often yield substantial improvement in measurement sensitivity per unit time. Availability of multiple receivers on the state-of-the-art commercial spectrometers allows spectra from different nuclear species to be recorded in parallel routinely. Experiments with multi-FID detection have been designed with both, homonuclear and multinuclear acquisition. We provide a brief overview of such techniques designed for applications in liquid- and solid- state NMR as well as in hyperpolarized samples. Here we show how these techniques have led to design of experiments that allow structure elucidation of small molecules and resonance assignment in proteins from a single measurement. Probes with multiple RF micro-coils routed to multiple NMR receivers provide an alternative way of increasing the throughput of modern NMR systems. Solid-state NMR experiments have also benefited immensely from both parallel and simultaneous FID acquisition in a variety of multi-dimensional pulse schemes. We believe that multi-FID detection will become an essential component of the future NMR methodologies effectively increasing the information content of NMR experiments and reducing the cost of NMR analysis.

Measuring strong one-bond dipolar couplings using REDOR in magic-angle spinning solid-state NMR.

Rotational-Echo DOuble Resonance, REDOR, is an experimentally robust and a well-established dipolar-recoupling technique to measure dipolar couplings between isolated pairs of spin-1/2 heteronuclei in solid-state nuclear magnetic resonance. REDOR can also be used to estimate motional order parameters when the bond distance is known, for example, in the case of directly bound nuclei. However, the relatively fast dipolar dephasing for strongly coupled spin-1/2 pairs, such as 13C-1H, makes the stroboscopic measurement required in this experiment challenging, even at fast Magic-Angle-Spinning (MAS) frequencies. In such cases, modified REDOR-based methods like Shifted-REDOR (S-REDOR) are used to scale the dipolar coupling compared to REDOR. This is achieved by changing the position of one of the two recoupling π-pulses in a rotor period. This feature, however, comes at the cost of mixing multiple Fourier components of the dipolar coupling and can, additionally, require high radio-frequency amplitudes to realise small scaling factors. We introduce here a general pulse scheme which involves shifting both the π pulses in the REDOR scheme to achieve arbitrary scaling factors whilst retaining the robustness and simplicity of REDOR recoupling and avoiding the disadvantages of S-REDOR. The classical REDOR is a specific case of this scheme with a scaling factor of one. We demonstrate the results on isolated 13C-15N and 1H-13C spin pairs at 20 and 62.5 kHz MAS, respectively.

Assignment of NMR resonances of protons covalently bound to photochemically active cofactors in photosynthetic reaction centers by 13C-1H photo-CIDNP MAS-J-HMQC experiment.

Modified versions of through-bond heteronuclear correlation (HETCOR) experiments are presented to take advantage of the light-induced hyperpolarization that occurs on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. Such 13C-1H photo-CIDNP MAS-J-HMQC and photo-CIDNP MAS-J-HSQC experiments are applied to acquire the 2D 13C-1H correlation spectra of selectively 13C-labeled photochemically active cofactors in the frozen quinone-blocked photosynthetic reaction center (RC) of the purple bacterium Rhodobacter (R.) sphaeroides wild-type (WT). Resulting spectra contain no correlation peaks arising from the protein backbone, which greatly simplifies the assignment of aliphatic region. Based on the photo-CIDNP MAS-J-HMQC NMR experiment, we obtained assignment of selective 1H NMR resonances of the cofactors involved in the electron transfer process in the RC and compared them with values theoretically predicted by density functional theory (DFT) calculation as well as with the chemical shifts obtained from monomeric cofactors in the solution. We also compared proton chemical shifts obtained by photo-CIDNP MAS-J-HMQC experiment under continuous illumination with the ones obtained in dark by classical cross-polarization (CP) HETCOR. We expect that the proposed approach will become a method of choice for obtaining 1H chemical shift maps of the active cofactors in photosynthetic RCs and will aid the interpretation of heteronuclear spin-torch experiments.