Pulsed electron spin nutation spectroscopy of weakly exchange-coupled biradicals: a general theoretical approach and determination of the spin dipolar interaction

Weakly exchange-coupled biradicals have attracted much attention in terms of their DNP application in NMR spectroscopy for biological systems or the use of synthetic electron-spin qubits. Pulse-ESR based electron spin nutation (ESN) spectroscopy applied to biradicals is generally treated as transition moment spectroscopy from the theoretical side, illustrating that it is a powerful and facile tool to determine relatively short distances between weakly exchange-coupled electron spins. The nutation frequency as a function of the microwave irradiation strength ω(1) (angular frequency) for any cases of weakly exchange-coupled systems can be classified into three categories; D(12) (spin dipolar interaction)-driven, Δg-driven and ω(1)-driven nutation behaviour with the increasing strength of ω(1). For hetero-spin biradicals, Δg effects can be a dominating characteristic in the biradical nutation spectroscopy. Two-dimensional pulse-based electron spin nutation (2D-ESN) spectroscopy operating at the X-band can afford to determine small values of D(12) in weakly exchange-coupled biradicals in rigid glasses. The analytical expressions derived here for ω(1)-dependent nutation frequencies are based on only four electronic spin states relevant to the biradicals, while real biradical systems often have sizable hyperfine interactions. Thus, we have evaluated nuclear hyperfine effects on the nutation frequencies to check the validity of the present theoretical treatment. The experimental spin dipolar coupling of a typical TEMPO-based biradical 1, (2,2,6,6-tetra[((2)H(3))methyl]-[3,3-(2)H(2),4-(2)H(1),5,5-(2)H(2)]piperidin-N-oxyl-4-yl)(2,2,6,6-tetra[((2)H(3))methyl]-[3,3-(2)H(2),4-(2)H(1),5,5-(2)H(2),(15)N]piperidin-(15)N-oxyl-4-yl) terephthalate in a toluene glass, with a distance of 1.69 nm between the two spin sites is D(12) = -32 MHz (the effect of the exchange coupling J(12) is vanishing due to the homo-spin sites of 1, i.e.Δg = 0), while 0 < |J(12)|≦ 1.0 MHz as determined by simulating the random-orientation CW ESR spectra of 1. In addition, we have carried out Q-band pulsed ELDOR (ELectron-electron DOuble Resonance) experiments to confirm whether the obtained values for D(12) and J(12) are accurate. The distance is in a fuzzy region for the distance-measurements capability of the conventional, powerful ELDOR spectroscopy. The strong and weak points of the ESN spectroscopy with a single microwave frequency applicable to weakly exchange-coupled multi-electron systems are discussed in comparison with conventional ELDOR spectroscopy. The theoretical spin dipolar tensor and exchange interaction of the TEMPO biradical, as obtained by sophisticated quantum chemical calculations, agree with the experimental ones.     

Anisotropic nuclear spin interactions for the morphology analysis of proteins in solution by NMR spectroscopy.

Determining the relative orientation of domains within a protein is an important problem in structural biology, which has been difficult to address by either X-ray crystallography or NMR. The structure of a multidomain protein in a crystal lattice can be altered by crystal packing forces, resulting in different domain arrangements from those in solution. On the other hand, conventional NMR primarily provides short-range structural information, including proton-proton distances derived from nuclear Overhauser effects (NOEs) and torsion angles through vicinal spin couplings. Thus, NMR cannot always determine the precise interdomain arrangements due to the sparsely observed spin interactions between domains. However, the weak alignment of proteins in solution has enabled a new NMR technique to determine the domain arrangement based on the different structural information, which defines the orientation of a structural unit in protein against the magnetic field. This technique relies on the anisotropic nuclear spin interactions that only occur for a molecule in a weakly aligned state. In this review, the basics of the new NMR approach are described with focusing on its application to domain orientation analysis. We also describe our recently established NMR approach using the same spin interactions, which expands the domain arrangement analysis to higher-molecular weight proteins over 100 kDa.     

Residual dipolar coupling measurements of transmembrane proteins using aligned low-q bicelles and high-resolution magic angle spinning NMR spectroscopy

Bicelles are a major medium form to produce weak alignment of soluble proteins for residual dipolar coupling (RDC) measurements. The obstacle to using the same type of bicelles for transmembrane proteins with solution-state NMR spectroscopy is the loss of signals due to the adhesion or penetration of the proteins into large bicelles, resulting in slow protein tumbling. In this study, weak alignment of the second and third transmembrane domains (TM23) of the human glycine receptor (GlyR) was achieved in low-q bicelles (q = DMPC/DHPC). Although protein-free bicelles with such low q would likely show isotropic properties, the insertion of TM23 induced weakly preferred orientations so that the RDC of the embedded protein can be measured. The extent of the alignment increased but the TM23 signal intensity decreased when q was varied from 0.19 to 0.60. A q of 0.50 was found to be an optimal compromise between alignment and the signal-to-noise ratio. In each pair of NMR experiments for RDC measurements, the same sample and pulse sequence were used, with one being performed at high-resolution magic-angle spinning to obtain pure J-couplings without RDC. A meaningful structure refinement in bicelles was possible by iteratively fitting the experimental RDCs to the back-calculated RDCs using the high-resolution NMR structure of GlyR TM23 in trifluoroethanol as the starting template. Combination of this method with the conventional high-resolution NMR in membrane mimicking mixtures of water and organic solvents offers an attractive way to derive structural information for membrane proteins in their native environment.     

Explicit treatment of spin labels in modeling of distance constraints from dipolar EPR and DEER

Current SDSL-EPR methods allow measurement of dipolar distances in the 8-70 A range; however, the use of extrinsic probes complicates the interpretation of these distances in modeling macromolecular structure and conformational changes. The data presented here show that interprobe distances correlate only weakly with Cbeta-Cbeta distances, especially for distances that are on the order of the spin label tether lengths. Explicitly incorporating the spin label into the modeling process increases the experiment/model correlation 4-fold and reduces the distance error from 6 A to 3 A.     

Paramagnetically induced residual dipolar couplings for solution structure determination of lanthanide binding proteins

Lanthanides may substitute calcium in calcium-binding proteins, such as, for instance, EF-hand proteins. Paramagnetic lanthanides are capable of orienting the protein in high magnetic fields to an extent similar to that obtained by using orienting devices, and each lanthanide orients according to its magnetic susceptibility tensor. Here, Ce(3+), Tb(3+), Dy(3+), Ho(3+), Er(3+), Tm(3+), Yb(3+) in the C-terminal site of calbindin D(9k) have been investigated. Such systems provide (1)H-(15)N residual dipolar couplings (rdc) which can be used for solution structure determinations. Within the frame of optimizing the use of residual dipolar couplings for efficient solution structure determination, it is proposed here to use a number of lanthanides (e.g., >2) to obtain the orientations of the internuclear vectors with respect to an arbitrary reference system. This is facilitated by the independent knowledge of the magnetic susceptibility anisotropy tensor of each metal, obtained from the analysis of the pseudocontact shifts. A further module of the program PARAMAGNETIC-DYANA, called RDCDYANA-ANGLES, is developed to efficiently incorporate such rdc-derived orientations, instead of the rdc themselves, as constraints in the solution structure calculation. This strategy is absolutely general and can be extended to any other pair of dipole-dipole coupled nuclei. The effect of mobility is also assessed. In principle, information on the mobility can be obtained with a number of lanthanide ions >5, or by combining a smaller number of lanthanide ions with a few orienting devices.     

Using measurements of anchoring energies of liquid crystals on surfaces to quantify proteins captured by immobilized ligands

We describe a simple optical method that employs measurement of the interaction energy of a liquid crystal (LC) with a surface (the so-called anchoring energy) to report proteins captured on surfaces through specific interactions with immobilized binding groups. To define the sensitivity and dynamic range of the response of the LC, we covalently immobilized a tyrosine-containing, 13-residue peptide sequence (Y1173) from the epidermal growth factor receptor/kinase (EGFR) at which autophosphorylation takes place and to which the adapter protein Shc binds. We determined that, on peptide-decorated (Y1173 or pY1173, where pY1173 is the corresponding phosphopeptide) surfaces incubated against anti-phosphotyrosine antibody, the anchoring energy of the LC decreased systematically from 4.4 to 1.4 microJ/m2 (with SEM=0.3 microJ/m2 for n=5) as the antibody concentration increased from 10 pM to 100 nM. Over the same range of antibody concentrations in solution and densities of immobilized peptides, independent ellipsometric measurements were not sufficiently sensitive to report the captured antibody (ellipsometric thicknesses were <0.1 nm). These results, when combined with control experiments reported in this article, provide the first demonstration of the use of anchoring energy measurements of LCs to report proteins captured by immobilized ligands on surfaces. The sensitivity and dynamic range of the methodology suggest that it may offer the basis of a simple yet broadly useful principle for reporting the interactions between proteins and other biomolecules that underlie complex and poorly understood chemical and biological events.     

Dipolar chemical shift correlation spectroscopy for homonuclear carbon distance measurements in proteins in the solid state: application to structure determination and refinement

High-resolution solid-state NMR spectroscopy has become a promising tool for protein structure determination. Here, we describe a new dipolar-chemical shift correlation experiment for the measurement of homonuclear 13C-13C distances in uniformly 13C,15N-labeled proteins and demonstrate its suitability for protein structure determination and refinement. The experiments were carried out on the beta1 immunoglobulin binding domain of protein G (GB1). Both intraresidue and interresidue distances between carbonyl atoms and atoms in the aliphatic side chains were collected using a three-dimensional chemical shift correlation spectroscopy experiment that uses homogeneously broadened rotational resonance recoupling for carbon mixing. A steady-state approximation for the polarization transfer function was employed in data analysis, and a total of 100 intramolecular distances were determined, all in the range 2.5-5.5 A. An additional 41 dipolar contacts were detected, but the corresponding distances could not be accurately quantified. Additional distance and torsional restraints were derived from the proton-driven spin diffusion measurements and from the chemical shift analysis, respectively. Using all these restraints, it was possible to refine the structure of GB1 to a root-mean square deviation of 0.8 A. The approach is of general applicability for peptides and small proteins and can be easily incorporated into a structure determination and refinement protocol.     

Noncovalent paramagnetic complexes: detection of halogen bonding in solution by ESR spectroscopy

Halogen-bonded complexes between aliphatic and aromatic iodoperfluorocarbons and persistent nitroxide radicals have been detected by ESR spectroscopy in solution. Quantitative data indicate that nitroxides behave as strong electron donors in halogen bonding, giving rise to interactions whose strength is close to that of strong hydrogen bonds. These results point to a novel design of supramolecular paramagnetic species.     

Pulsed pressure perturbations, an extra dimension in NMR spectroscopy of proteins

The introduction of multidimensional NMR spectroscopy was a breakthrough in biological NMR methodology because it allowed the unequivocal correlation of different spin states of the system. The introduction of large pressure perturbations in the corresponding radio frequency (RF) pulse sequences adds an extra structural dimension into these experiments. We have developed a microprocessor-controlled pressure jump unit that is able to introduce fast, strong pressure changes at any point in the pulse sequences. Repetitive pressure changes of 80 MPa in the sample tube are thus feasible in less than 30 ms. Two general forms of these experiments are proposed here, the pressure perturbation transient state spectroscopy (PPTSS) and the pressure perturbation state correlation spectroscopy (PPSCS). PPTSS can be used to measure the rate constants and the activation energies and activation volumes for the transition between different conformational states including the folded and unfolded state of proteins, for polymerization-depolymerization processes, and for ligand binding at atomic resolution. PPSCS spectroscopy correlates the NMR parameters of different pressure-induced states of the system, thus allowing the measurement of properties of a given pressure induced state such as a folding intermediate in a different state, for example, the folded state. Selected examples for PPTSS and PPSCS spectroscopy are presented in this Article.     

Investigation of nonspecific effects of different dyes in the screening of labeled carbohydrates against immobilized proteins

[structure: see text] Carbohydrates play an important role in life processes, and combinatorial chemistry can provide useful sources of thousands of synthetic carbohydrates as potential ligands for biological receptors. To accelerate the detection of positive hits arising from specific interactions between a carbohydrate and a protein, the use of fluorescent dyes can serve as a reliable detecting tool. A study of labeled carbohydrates to lectins conjugated to a solid-support shows that succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X) dye provides by far the lowest level of nonspecific interaction with immobilized protein. This observation is in stark contrast with the commonly used labeling reagents constituted of charged and aromatic groups, for instance, FITC and TAMRA dyes.     

Accurate heteronuclear distance measurements at all magic-angle spinning frequencies in solid-state NMR spectroscopy

Heteronuclear dipolar coupling is indispensable in revealing vital information related to the molecular structure and dynamics, as well as intermolecular interactions in various solid materials. Although numerous approaches have been developed to selectively reintroduce heteronuclear dipolar coupling under MAS, most of them lack universality and can only be applied to limited spin systems. Herein, we introduce a new and robust technique dubbed phase modulated rotary resonance (PMRR) for reintroducing heteronuclear dipolar couplings while suppressing all other interactions under a broad range of MAS conditions. The standard PMRR requires the radiofrequency (RF) field strength of only twice the MAS frequency, can efficiently recouple the dipolar couplings with a large scaling factor of 0.50, and is robust to experimental imperfections. Moreover, the adjustable window modification of PMRR, dubbed wPMRR, can improve its performance remarkably, making it well suited for the accurate determination of dipolar couplings in various spin systems. The robust performance of such pulse sequences has been verified theoretically and experimentally via model compounds, at different MAS frequencies. The application of the PMRR technique was demonstrated on the H-ZSM-5 zeolite, where the interaction between the Brønsted acidic hydroxyl groups of H-ZSM-5 and the absorbed trimethylphosphine oxide (TMPO) were probed, revealing the detailed configuration of super acid sites.

A new and robust technique dubbed phase modulated rotary resonance (PMRR) was proposed for the accurate determination of heteronuclear dipolar coupling under a broad range of MAS conditions in solid-state NMR spectroscopy.     

Reusable Pd nanoparticles immobilized on functional ionic liquid co-polymerized with styrene for Suzuki reactions in water-ethanol solution

Co-polymer of 3-(2,3-dihydroxypropyl)-1-vinylimidazolium chloride and styrene was synthesized and used as a support of Pd nanoparticles. The Pd@poly-Sty-co-diOH-Cl catalyst can efficiently catalyze Suzuki reactions for a wide range of aryl iodides and bromides with 0.05 mol % Pd at 70 °C in water-ethanol solution under air, and the catalyst can be recycled and reused for several times without significant loss of activity.     

High-sensitivity observation of dipolar exchange and NOEs between exchangeable protons in proteins by 3D solid-state NMR spectroscopy

A highly sensitive new 1H-detected 3D solid-state NMR method is described for characterizing 1H-1H spin exchange in nanocrystalline samples of 15N- and 2H-enriched protein. Long-range contacts are observed in human ubiquitin. The method is also used to show that numerous NOEs between backbone amides and crystal water protons can be observed.