Direct evidence for a deprotonated lysine serving as a H-bond "acceptor" in a photoreceptor protein.

Deprotonation or suppression of the pKa of the amino group of a lysine sidechain is a widely recognized phenomenon whereby the sidechain amino group transiently can act as a nucleophile at the active site of enzymatic reactions. However, a deprotonated lysine and its molecular interactions have not been directly experimentally detected. Here, we demonstrate a deprotonated lysine stably serving as an "acceptor" in a H-bond between the photosensor protein RcaE and its chromophore. Signal splitting and trans-H-bond J coupling observed by NMR spectroscopy provide direct evidence that Lys261 is deprotonated and serves as a H-bond acceptor for the chromophore NH group. Quantum mechanical/molecular mechanical calculations also indicate that this H-bond exists stably. Interestingly, the sidechain amino group of the lysine can act as both donor and acceptor. The remarkable shift in the H-bond characteristics arises from a decrease in solvation, triggered by photoisomerization. Our results provide insights into the dual role of this lysine. This mechanism has broad implications for other biological reactions in which lysine plays a role.

Single-Shot MRI in parahydrogen hyperpolarized samples.

The site-specific signal enhancement provided by parahydrogen induced polarization (PHIP) may be combined with magnetic resonance imaging (MRI) to study chemical and biomolecular processes. However, imaging of hydrogen nuclei (1H) is hampered by background signals arising from the presence of thermally polarized nuclei. Additionally, fast imaging sequences are commonly based on multiple radio-frequency pulses, where the signals resulting from PHIP oscillate due to the evolution with a J-coupling Hamiltonian. In this article, an innovative imaging scheme for single-scan MRI is presented that effectively detects hyperpolarized components while simultaneously canceling out thermal contributions. This method is based on the quenching of inherent oscillations of PHIP-originated signals due to J-couplings during the multipulse sequence and the suppression of thermal signals by spin dynamics and a tailored restructuring of the k-space. A series of numerical simulations on specific two- and three-spin systems serve to support the feasibility of the approach. Furthermore, this theoretical study demonstrates the potential of combining hyperpolarization and long-lived states (PHIP and LLS) in the selected molecules, which could be seen as a preliminary step towards the development of fast imaging techniques, for example in the field of biomolecular research.

Through-space scalar spin-spin coupling: from rigid intramolecular cases to short-lived van der Waals complexes.

We will discuss, with the help of few selected examples, how the concept of through-space scalar spin-spin coupling between non covalently bonded nuclei has evolved in recent years. We will first present systems where 'no covalent bond' actually means that the two atoms are separated by a large number of bonds; then we will see cases where it is referred to true van der Waals dimers, but with the two atoms somehow constrained in their positions; we will finish with the most recent examples of liquids and even gaseous mixtures with full translational degrees of freedom in a regime of intermolecular/interatomic fast exchange.

Echo time optimization for in-vivo measurement of unsaturated lipid resonances using J-difference-edited MRS.

PURPOSE: Measuring lipid composition provides more information than just total lipid content. Hence, the non-invasive measurement of unsaturated lipid protons with both high efficiency and precision is of pressing need. This study was to optimize echo time (TE) for the best resolving of J-difference editing of unsaturated lipid resonances. METHODS: The TE dependence of J-difference-edited (JDE) MRS was verified in the density-matrix simulation, soybean oil phantom, in-vivo experiments of white adipose tissue (WAT), and skeletal muscles using single-voxel MEGA-PRESS sequence at 3T. The peak SNRs and Cramér-Rao lower bounds (CRLBs) acquired at the proposed TE of 45 ms and previously published TE of 70 ms were compared (eight pairs) in WAT, extramyocelluar lipids (EMCLs), and intramyocellular lipids (IMCLs). The lipid composition in skeletal muscles was compared between healthy males (n = 7) and females (n = 7). RESULTS: The optimal TE was suggested as 45 ms. Compared to 70 ms, the mean signal gains at TE of 45 ms were 151% in WAT, 168% in EMCL, 204% in IMCL for allylic resonance, and 52% in EMCL for diallylic resonance. CRLBs were significantly reduced at TE of 45 ms in WAT, EMCL, IMCL for allylic resonance and in EMCL for diallylic resonance. With TE of 45 ms, significant gender differences were found in the lipid composition in EMCL pools, while no difference in IMCL pools. CONCLUSION: The JDE-MRS protocol with TE of 45 ms allows improved quantification of unsaturated lipid resonances in vivo and future lipid metabolism investigations.

Indirect Nuclear Spin-Spin Exchange Coupling through Solvated Electron in Small Sizes of Cyclic and Cage-Shaped Perfluoroalkanes.

Small perfluorocycloalkanes (hexafluorocyclopropane (c-C3 F6 ), octafluorocyclobutane (c-C4 F8 ) and decafluorocyclopentane (c-C5 F10 ) and cage-shaped perfluoroalkanes (perfluoro tetrahedral alkane (C4 F4 ), perfluoro prismane (C6 F6 ) and perfluoro cubane (C8 F8 )) are better electron scavengers. The captured excess electrons are weakly bound inside their backbone voids or over their backbones, forming the solvated electron ( e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ ) systems (e@c-Cn F2n s (n=3, 4, 5) and e@Cn Fn (n=4, 6, 8)). There have been many studies on the structures and properties of such e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ systems. However, the effect of e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ on the indirect nuclear spin-spin coupling (J-coupling) is unknown. In this work, we explore how e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ affects Ne J-coupling between two coupled F nuclei (Ne JFF -coupling) in perfluoroalkane e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ systems through density functional theory calculations. We find unusual trans-Ne JFF -couplings (two coupled F nuclei in trans-position) in e@c-Cn F2n (n=3, 4, 5) and Ne JFF -couplings in e@Cn Fn (n=4, 6, 8). One excess electron not only changes the molecular structures, but also enforces unique distributions and properties, depending on the structural characteristics. We also confirm that such unusual Ne JFF -couplings are realized by through- e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ (T-SE) transmission mechanism, rather than the conventional through-bonds (T-B)/through-space (T-S) ones. The novel transmission mechanism consists of the T-SE coupling path (path 1) and e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ -enhanced T-B ⊕ ${ \oplus }$ T-S coupling path (path 2), and the two paths jointly control Ne JFF through cooperation and competition. Interestingly, the former plays a dominant role for long-range Ne JFF -coupling (N=5), while the latter plays a role in the short-range Ne JFF -coupling (N=3, 4). Path bending angle mainly influences path 1, while path 2 is mainly influenced by the path length. This work not only provides novel insights into the mediating role of e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ in the coupling information exchange, but also proposes a new e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ -based coupling mechanism, possibly opening up potential applications for the e sol - ${{\rm{e}}_{{\rm{sol}}}^- }$ -based indirect nuclear spin couplings.

Homonuclear J-coupling spectroscopy using J-synchronized echo detection.

In the strong coupling regime with J-coupling much larger than chemical shift differences, J-coupling spectroscopy enables spectral identification of molecules even when conventional NMR fails. While this classically required the presence of a heteronucleus, we recently showed that J-coupling spectra can be acquired in many homonuclear systems using spin-lock induced crossing (SLIC). Here, we present an alternative method using a spin echo train in lieu of a spin-locking SLIC pulse, which has a number of advantages. In particular, spin echo acquisition within the pulse train enables simultaneous collection of time and frequency data. The resulting 2D spectrum can be used to study dynamic spin evolution, and the time domain data can be averaged to create a 1D J-coupling spectrum with increased signal-to-noise ratio.

Exploring Nearest Neighbor Interactions and Their Influence on the Gibbs Energy Landscape of Unfolded Proteins and Peptides.

The Flory isolated pair hypothesis (IPH) is one of the corner stones of the random coil model, which is generally invoked to describe the conformational dynamics of unfolded and intrinsically disordered proteins (IDPs). It stipulates, that individual residues sample the entire sterically allowed space of the Ramachandran plot without exhibiting any correlations with the conformational dynamics of its neighbors. However, multiple lines of computational, bioinformatic and experimental evidence suggest that nearest neighbors have a significant influence on the conformational sampling of amino acid residues. This implies that the conformational entropy of unfolded polypeptides and proteins is much less than one would expect based on the Ramachandran plots of individual residues. A further implication is that the Gibbs energies of residues in unfolded proteins or polypeptides are not additive. This review provides an overview of what is currently known and what has yet to be explored regarding nearest neighbor interactions in unfolded proteins.

Using spectrally-selective radiofrequency pulses to enhance lactate signal for diffusion-weighted MRS measurements in vivo.

Measurement of lactate diffusion properties using diffusion-weighted magnetic resonance spectroscopy in vivo may allow elucidating brain lactate cellular compartmentation, which would be of great importance for neuroscience. However, measuring lactate signal is complicated by low signal-to-noise ratio due to low lactate concentration and J-modulation of its 1.3 ppm peak. In this work, we assess the benefits of using a diffusion-weighting spin echo block and spectrally selective refocusing pulses to suppress the effect of J-coupling on the 1.3 ppm lactate resonance, by not refocusing its coupling partner at 4.1 ppm. Two different kinds of spectrally selective pulses, either polychromatic or single-band, are evaluated in the mouse brain at 11.7 T. Almost complete suppression of J-modulation is shown, resulting in an approximately two-fold signal increase as compared to a reference STE-LASER sequence (for the specific diffusion times used in this work). Repeated measurements confirm that lactate diffusion-weighted signal attenuation is measured with an approximately two-fold precision.

Fat unsaturation measures in tibial, subcutaneous and breast adipose tissue using short and long TE MRS at 3 T.

Fat unsaturation and poly-unsaturation measures can be obtained in vivo with magnetic resonance spectroscopy (MRS) through the olefinic (≈5.4 ppm) and diallylic (≈2.8 ppm) resonances, respectively. Long echo time (TE) MRS sequences have been previously optimized for olefinic/methylene (≈1.3 ppm) or olefinic/methyl (≈0.9 ppm) measures. The objectives of this work, using a Point RESolved Spectroscopy (PRESS) sequence, are to: 1) Investigate olefinic, methyl and methylene resonance decay in subcutaneous, tibial, and breast adipose tissue to determine if a direct comparison of unsaturation measures can be made without correction for T2 losses. 2) Assess intra-individual fat unsaturation and poly-unsaturation measures in the three adipose tissues. 3) Estimate correction factors for olefinic to methylene ratios to compensate for J-coupling and T2 relaxation losses that take place when increasing PRESS TE from 40 ms to 200 ms (previously optimized long-TE). 4) Investigate the utility of an inversion recovery for resolving the olefinic resonance from water in adipose tissue. PRESS spectra were acquired from the three adipose regions (breast in female only) in healthy volunteers at 3 T. It was found that olefinic and methyl signal decays faster in breast tissue compared to in tibial bone marrow. Poly-unsaturation measures (diallylic/methylene) differ for tibial bone marrow compared to subcutaneous and breast adipose tissue, with average values of 1.7 ± 0.4, 2.2 ± 0.4, and 2.3 ± 0.8%, respectively. PRESS (TE = 40 ms) with an inversion recovery resolves the olefinic and water resonances in breast tissue with a signal to noise ratio approximately six times greater than that using PRESS with a TE of 200 ms. Stimulated Echo Acquisition Mode (STEAM) with a TE of 20 ms (mixing time of 20 ms) was also combined with IR to resolve the olefinic resonance from that of water is spinal bone marrow.

Probing 29Si-17O connectivities and proximities by solid-state NMR.

The measurement of dipolar and J- couplings between 29Si and 17O isotopes is challenging owing to (i) the low abundance of both isotopes and (ii) their close Larmor frequencies, which only differ by 19%. These issues are circumvented here by the use of isotopic enrichment and dedicated triple-resonance magic-angle spinning NMR probe. The surface of 29Si-enriched silica was labelled with 17O isotope and heated at 80 and 200 °C. 29Si-17O connectivities and proximities were probed using two-dimensional (2D) through-bond and through-space heteronuclear multiple-quantum coherences (J- and D-HMQC) experiments between 17O and 29Si nuclei. The simulation of the build-up of the J- and D-HMQC signals allowed the first experimental measurement of J- and dipolar coupling constants between 17O and 29Si nuclei. These HMQC experiments allow distinguishing two distinct siloxane (SiOSi) oxygen sites: (i) those covalently bonded to Q3 and Q4 groups, having a hydroxyl group as a second neighbour and (ii) those covalently bonded to two Q4 groups. The measured J- and dipolar coupling constants of siloxane 17O nucleus with Q4 29Si nuclei differ from those with Q3 29Si nuclei. These results indicate that the 29Si-17O one-bond J-coupling and Si-O bond length depend on the second neighbours of the Si atoms.

Homonuclear J-Coupling Spectroscopy at Low Magnetic Fields using Spin-Lock Induced Crossing*.

Nuclear magnetic resonance (NMR) spectroscopy usually requires high magnetic fields to create spectral resolution among different proton species. Although proton signals can also be detected at low fields the spectrum exhibits a single line if J-coupling is stronger than chemical shift dispersion. In this work, we demonstrate that the spectra can nevertheless be acquired in this strong-coupling regime using a novel pulse sequence called spin-lock induced crossing (SLIC). This techniques probes energy level crossings induced by a weak spin-locking pulse and produces a unique J-coupling spectrum for most organic molecules. Unlike other forms of low-field J-coupling spectroscopy, our technique does not require the presence of heteronuclei and can be used for most compounds in their native state. We performed SLIC spectroscopy on a number of small molecules at 276 kHz and 20.8 MHZ and show that the simulated SLIC spectra agree well with measurements.

Discrimination of PHIP Signals Through their Evolution in Multipulse Sequences.

The antiphase character of the PHIP associated signals after a hydrogenation reaction is particularly sensitive to line broadening introduced by magnetic field inhomogeneities and interferences by the presence of resonance lines steaming from a large amount of thermally polarized spins. These obstacles impose a limitation in the detection of reaction products as well as in the experimental setups. A simple way to overcome these impediments consists of acquiring the signal with a train of refocusing pulses instead of a single r.f. pulse. We present here a number of examples where this multipulse acquisition, denominated PhD-PHIP, displays its potentiality in improving the information related to hyperpolarized spins performed in a sample, where the former parahydrogen nuclei are part of a complex J-coupling network.

Isolation of diverse bioactive compounds from Euphorbia balsamifera: Cytotoxicity and antibacterial activity studies.

Antibacterial and cytotoxic activities of Euphorbia balsamifera, fractions and pure compounds were evaluated. The cytotoxic assays for HCT116, HePG2 and MCF7 showed a significant IC50: 54.7 and 76.2 µg/mL of non-polar fraction "n-hexane" against HCT116 and HePG2, respectively. Antibacterial results revealed that plant fractions exhibited significant potential against the tested pathogens than the total extract where n-butanol and ethyl acetate fractions showed significant antibacterial activity (P < 0.05) against tested bacterial strains. Isolation and structure determination of compounds from n-hexane and n-butanol fractions were performed. From n-hexane fraction, 29-nor-cycloartanol (1), lanost-8-en-3-ol (2a), cycloartanol (2b) and kampferol-3,4'-dimethyl ether (3) were isolated and structurally identified, along with 24 compounds were tentatively identified by GC-MS. From the polar n-butanol fraction, 4-O-ß-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (4), 4-O-α-L-rhamnosyl-(1 â†’ 6)-ß-D-glucopyranosyl-2-hydroxy-6methoxy-acetophenone (5), quercetin-3-O-glucopyranoside (6) and isoorientin (7) were assigned. Structures of the obtained compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Except compounds 1 and 5, all reported compounds announced antibacterial efficiency. Compound 2 showed selectively the highest activity against Enterococcus faecalis (22 ± 0.13 mm), meanwhile 4-O-ß-D-glucopyranosyl-2-hydroxy-6-methoxyacetophenone (4) showed broadly the highest antibacterial activity with MIC of 1.15-1.88 mg/mL against the test Gram-positive and Gram-negative bacteria. Cytotoxic assays indicated that kampferol-3,4'-dimethyl ether (3) exhibited the highest activity with matching IC50 values to doxorubicin; 111.46, 42.67 and 44.90 µM against HCT116, HePG2 and MCF7, respectively, however, it is toxic on retina normal cell line RPE1.

Radiofrequency encoded Only Parahydrogen SpectroscopY.

A new pulse sequence aimed to filter out NMR signals coming from thermally polarized protons in PHIP experiments based on the OPSY pulse sequence (Only Parahydrogen SpectroscopY) is presented. In analogy to OPSY, which removes thermal polarization by using a pair of magnetic field gradient pulses with an intensity ratio 1:2 and equal duration, the same effect can be achieved using inhomogeneous radiofrequency fields. The spatial dependence of the radiofrequency field is used to control the Hamiltonian, which results in an effective suppression of thermal contributions in the NMR signal, while PHIP originated signals remain unmodified. A theoretical model for the radiofrequency encoded only parahydrogen (REOPSY) sequence is presented along with an experimental implementation on a birdcage coil in a 7 T magnetic field. The control level achieved by this strategy allows the inclusion of a long train of refocusing pulses. Therefore, the new sequence can be combined with the parahydrogen discriminated PHIP (PhD-PHIP) pulse sequence as a detection block to improve sensitivity and resolution in a single-scan experiment. Experiments with REOPSY and REOPSY+PhD-PHIP are presented in thermally and hyperpolarized samples.

Quantum mechanical reference spectrum simulation for precursors and degradation products of chemicals relevant to the Chemical Weapons Convention.

A selection of acidic, alkaline and neutral degradation products relevant to the Chemical Weapons Convention was studied in wide range of pH conditions to determine their spin systems as well as spectral parameters. The pH dependence of chemical shifts and J couplings was parameterized using Henderson-Hasselbalch-based functions using dichloromethane as additional shift reference in TSP-d4 referenced spectra. The resulting parameters allowed calculation of precise chemical shifts and J coupling constants in arbitrary pH conditions. The validity of the obtained spin system definitions and parameters as a source of quantum mechanically simulated reference data in chemical verification analysis is demonstrated.

MRI of [2-13 C]Lactate without J-coupling artifacts.

PURPOSE: Imaging of [2-13 C]lactate, a metabolic product of [2-13 C]pyruvate, is over considerable interest in hyperpolarized 13 C studies. However, artifact-free imaging of a J-coupled nuclear spin species can be challenging due to the peak-splitting induced by the spin-spin interactions. In this work, two new techniques resolving these J-modulated artifacts are presented. THEORY AND METHODS: The Product Operator Formalism (POF) of density matrix theory is used to both numerically and analytically derive the coherences arising during radiofrequency excitation and readout of a J-coupled spin system. A combination of computer simulations and experiments with [2-13 C]lactate and 13 C-formate phantoms are then used to verify the performance of two imaging methods. In the first approach, a quadrature imaging technique is used to eliminate scalar coupling artifacts via the combination of in-phase and quadrature images acquired at echo times differing by 1/2J with an echoplanar readout. The second approach employs a highly narrowband RF excitation pulse to image a single peak from the J-coupled doublet. RESULTS: Simulations using a numerical Shepp-Logan phantom, in vitro experiments using thermally polarized [2-13 C]lactate, thermally and hyperpolarized 13 C-formate phantoms, and in vivo imaging of [2-13 C]lactate produced in rat brain following injection of hyperpolarized [2-13 C]pyruvate show artifact-free images and demonstrate potential utility of these methods. CONCLUSION: The quadrature imaging and the narrowband excitation techniques resolve the J-coupling induced ghosting and blurring artifacts present with conventional MRI of J-coupled signals such as [2-13 C]lactate.

Magnetic resonance Spectrum simulator (MARSS), a novel software package for fast and computationally efficient basis set simulation.

The aim of this study was to develop a novel software platform for the simulation of magnetic resonance spin systems, capable of simulating a large number of spatial points (1283 ) for large in vivo spin systems (up to seven coupled spins) in a time frame of the order of a few minutes. The quantum mechanical density-matrix formalism is applied, a coherence pathway filter is utilized for handling unwanted coherence pathways, and the 1D projection method, which provides a substantial reduction in computation time for a large number of spatial points, is extended to include sequences of an arbitrary number of RF pulses. The novel software package, written in MATLAB, computes a basis set of 23 different metabolites (including the two anomers of glucose, seven coupled spins) with 1283 spatial points in 26 min for a three-pulse experiment on a personal desktop computer. The simulated spectra are experimentally verified with data from both phantom and in vivo MEGA-sLASER experiments. Recommendations are provided regarding the various assumptions made when computing a basis set for in vivo MRS with respect to the number of spatial points simulated and the consideration of relaxation.

Diffusion measurements with continuous hydrogenation in PHIP.

DOSY is a powerful spectroscopic NMR technique that resolves components in mixtures through the evaluation of different diffusion coefficients. The application of DOSY to dilute mixtures is hampered by the low signal to noise ratios (SNR), leading to long acquisition times. The use of PHIP may resolve this issue as long as reproducible signals are obtained in order to perform 2D experiments. Here we show that the use of hollow membranes and adequate gas flow produce constant polarization for a time-span that enables the acquisition of 2D experiments. A pressure gradient is evidenced by the presence of convection, which is accounted for by using a DPGSE sequence. The influence of J-coupling evolution during the sequence is studied both numerically and experimentally, to determine the optimum echo-time. The applicability of the method for samples with poor SNR is explored by setting the reaction rate to achieve a low intensity of polarized signals.

Revealing weak histidine 15N homonuclear scalar couplings using Solid-State Magic-Angle-Spinning NMR spectroscopy.

The tautomeric structure and chemistry of the histidine imidazole ring play active roles in many structurally and functionally important proteins and polypeptides. While in NMR spectroscopy histidine chemical shifts (e.g. 15N, 13C, and 1H) have been commonly used to characterize the tautomeric structure, hydrogen bonding, and torsion angles, homonuclear 15N scalar couplings in histidine have rarely been reported. Here, we propose double spin-echo sequences to compare the observed signals with and without a 90° pulse between the two spin-echo periods, such that their signal ratio as a function of the echo time solely depends on homonuclear scalar couplings, allowing for measuring weak homonuclear scalar couplings without influence from transverse dephasing effects, thus capable of revealing hydrogen-bond mediated 15N-15N J-couplings that can provide direct and definitive evidence for the formation of N…H…N hydrogen-bonding associated with the imidazole ring. We used two 13C,15N labeled histidine samples recrystallized from solutions at pH 6.3 and pH 11.0 to demonstrate the feasibility of this method and reveal the existence of a weak two-bond scalar coupling between the Nδ1 and Nε2 sites in the histidine imidazole ring in three tautomeric states and the presence of a hydrogen-bond mediated scalar coupling between the Nδ1 site in the imidazole ring and the backbone Nα site in the histidine neutral τ and π states. Our results demonstrate that weak 15N homonuclear scalar couplings can be measured even when their values are less than their corresponding intrinsic natural linewidths, thus providing direct and definitive evidence for the formation of N…H…N hydrogen bonding that is associated with the histidine imidazole ring.

Understanding and solving abnormal peak splitting in 3D HCCH-TOCSY and HCC(CO)NH-TOCSY.

The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum information out of challenging biomolecules. Here we investigate the source of and propose solutions for abnormal peak splitting ranging from 152 to 80 Hz and below that were found in three popular TOCSY-based experiment types: H(F1)-C(F2)-DIPSI-H(F3), C(F1)-DIPSI-C(F2)-H(F3), and C(F1)-DIPSI-N(F2)-HN(F3). Peak splitting occurs in the indirect C(F1) or C(F2) dimension before DIPSI and analyses indicate that the artifacts are resulted mainly from the DIPSI transforming a double spin order [Formula: see text] from 13C-13C scalar 1JCC coupling during t1 into observable megnetization. The splitting is recapitulated by numerical simulation and approaches are proposed to remove it. Adding a pure delay of 3.7 ms immediately before DIPSI is a simple and effective strategy to achieve 3D HCCH-TOCSY and HCC(CO)NH-TOCSY spectra free of splitting with full crosspeak intensity.