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1.
Phys Chem Chem Phys ; 24(39): 24238-24245, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36168981

RESUMO

Nuclear spin relaxation mechanisms are often difficult to isolate and identify, especially in molecules with internal flexibility. Here we combine experimental work with computation in order to determine the major mechanisms responsible for 31P spin-lattice and singlet order (SO) relaxation in pyrophosphate, a physiologically relevant molecule. Using field-shuttling relaxation measurements (from 2 µT to 9.4 T) and rates calculated from molecular dynamics (MD) trajectories, we identified chemical shift anisotropy (CSA) and spin-rotation as the major mechanisms, with minor contributions from intra- and intermolecular coupling. The significant spin-rotation interaction is a consequence of the relatively rapid rotation of the -PO32- entities around the bridging P-O bonds, and is treated by a combination of MD simulations and quantum chemistry calculations. Spin-lattice relaxation was predicted well without adjustable parameters, and for SO relaxation one parameter was extracted from the comparison between experiment and computation (a correlation coefficient between the rotational motion of the groups).


Assuntos
Difosfatos , Simulação de Dinâmica Molecular , Anisotropia , Espectroscopia de Ressonância Magnética
2.
J Chem Phys ; 157(13): 134302, 2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36208995

RESUMO

Coupled pairs of spin-1/2 nuclei support one singlet state and three triplet states. In many circumstances, the nuclear singlet order, defined as the difference between the singlet population and the mean of the triplet populations, is a long-lived state that persists for a relatively long time in solution. Various methods have been proposed for generating singlet order, starting from nuclear magnetization. This requires the stimulation of singlet-to-triplet transitions by modulated radiofrequency fields. We show that a recently described pulse sequence, known as PulsePol [Schwartz et al., Sci. Adv., 4, eaat8978 (2018)], is an efficient technique for converting magnetization into long-lived singlet order. We show that the operation of this pulse sequence may be understood by adapting the theory of symmetry-based recoupling sequences in magic-angle-spinning solid-state nuclear magnetic resonance (NMR). The concept of riffling allows PulsePol to be interpreted by using the theory of symmetry-based pulse sequences and explains its robustness. This theory is used to derive a range of new pulse sequences for performing singlet-triplet excitation and conversion in solution NMR. Schemes for further enhancing the robustness of the transformations are demonstrated.

3.
J Labelled Comp Radiopharm ; 65(14): 361-368, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36272110

RESUMO

A synthesis of N-monodeuteriomethyl-2-substituted piperidines is described. An efficient and readily scalable anodic methoxylation of N-formylpiperidine in an undivided microfluidic electrolysis cell delivers methoxylated piperidine 3, which is a precursor to a N-formyliminium ion and enables C-nucleophiles to be introduced at the 2-position. The isotopically labelled N-deuteriomethyl group is installed using the Eschweiler-Clarke reaction with formic acid-d2 and unlabelled formaldehyde. Monodeuterated N-methyl groups in these molecular systems possess small isotropic proton chemical shift differences important in the investigation of molecules that are able to support long-lived nuclear spin states in solution nuclear magnetic resonance.


Assuntos
Piperidinas , Eletroquímica , Piperidinas/química
4.
Phys Chem Chem Phys ; 23(16): 9851-9859, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33908503

RESUMO

Measurements of singlet spin order decay rates are time consuming due to the long-lived nature of this form of order and the typical pseudo-2D mode of acquisition. Additionally, this acquisition modality is not ideal for experiments run on hyperpolarized order because of the single-shot nature of hyperpolarization techniques. We present a methodology based on spatial encoding that not only significantly reduces the duration of these experiments but also confers compatibility using spin hyperpolarization techniques. The method condenses in a single shot the variable delay array used to measure decay rates in conventional pseudo-2D relaxation experiments. This results in a substantial time saving factor and, more importantly, makes the experiment compatible with hyperpolarization techniques since only a single hyperpolarized sample is required. Furthermore, the presented method, besides offering savings on time and costs, avoids reproducibility concerns associated with repetition in the hyperpolarization procedure. The method accelerates the measurement and characterization of singlet order decay times, and, when coupled with hyperpolarization techniques, can facilitate the quest for systems with very long decay times.

5.
Angew Chem Int Ed Engl ; 60(30): 16442-16447, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-33973334

RESUMO

Channelrhodopsin-2 (ChR2) is a light-gated cation channel and was used to lay the foundations of optogenetics. Its dark state X-ray structure has been determined in 2017 for the wild-type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP-enhanced solid-state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle.


Assuntos
Channelrhodopsins/química , Chlamydomonas reinhardtii/química , Diterpenos/química , Retinaldeído/química , Bases de Schiff/química , Cátions/química , Luz , Espectroscopia de Ressonância Magnética , Processos Fotoquímicos , Fótons , Conformação Proteica
6.
J Chem Phys ; 152(16): 164201, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32357786

RESUMO

Algorithmic cooling methods manipulate an open quantum system in order to lower its temperature below that of the environment. We achieve significant cooling of an ensemble of nuclear spin-pair systems by exploiting the long-lived nuclear singlet state, which is an antisymmetric quantum superposition of the "up" and "down" Zeeman states. The effect is demonstrated by nuclear magnetic resonance experiments on a molecular system containing a coupled pair of near-equivalent 13C nuclei. The populations of the system are subjected to a repeating sequence of cyclic permutations separated by relaxation intervals. The long-lived nuclear singlet order is pumped well beyond the unitary limit. The pumped singlet order is converted into nuclear magnetization which is enhanced by 21% relative to its thermal equilibrium value.

7.
J Struct Biol ; 206(1): 55-65, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29879487

RESUMO

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.


Assuntos
Proteínas de Bactérias/genética , Flavobacteriaceae/genética , Espectroscopia de Ressonância Magnética/métodos , Mutação , Rodopsinas Microbianas/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flavobacteriaceae/metabolismo , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo
8.
J Am Chem Soc ; 141(50): 19888-19901, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31756090

RESUMO

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.

9.
Phys Chem Chem Phys ; 21(11): 6087-6100, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30810569

RESUMO

We present approaches for an efficient excitation of singlet-triplet coherences in pairs of nearly-equivalent spins. Standard Nuclear Magnetic Resonance (NMR) pulse sequences do not excite these coherences at all or with very low efficiency. The single quantum singlet-triplet coherences, here termed the outer singlet-triplet coherences, correspond to lines of low intensity in the NMR spectrum of a strongly-coupled spin pair (they are sometimes referred to as "forbidden transitions"), whereas the zero-quantum coherences, here termed the inner singlet-triplet coherences, do not have a direct spectral manifestation. In the present study, we investigated singlet-triplet coherences in a pair of nearly-equivalent carbon spins of the 13C-isotopomer of a specially designed naphthalene derivative with optimized relaxation properties. We propose and compare several techniques to drive the singlet-triplet coherence in strongly coupled spin pairs. First, we study different methods for efficient excitation of the outer singlet-triplet coherences. The achieved conversion efficiency of magnetization to the coherences of interest is close to the theoretically allowed maximum. Second, we propose methods to convert the outer coherences into the inner singlet-triplet coherence. The inner singlet-triplet coherence is insensitive to field inhomogeneity and can be long-lived. By probing this coherence, we perform a very precise measurement of the spin-spin J-couplings. A remarkable property of this coherence is that it can be preserved even in absence of a spin-locking radiofrequency field. Consequently, it is possible to shuttle the sample between different magnetic fields preserving the coherence. This allows one to study the field dependence of the relaxation time, TIST, of the inner singlet-triplet coherence by performing field-cycling experiments. We observed dramatic changes of the ratio TIST/T1 from about 1 (in strong fields) up to 2.4 (in weak fields), which is the evidence of a significant influence of the chemical shift anisotropy on relaxation. We have detected a remarkably long lifetime of the inner singlet-triplet coherence of about 200 s at the magnetic field of 5 mT.

10.
J Chem Phys ; 151(23): 234203, 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864263

RESUMO

Some nuclear spin systems support long-lived states, which display greatly extended relaxation times relative to the relaxation time of nuclear spin magnetization. In spin-1/2 pairs, such a long-lived state is given by singlet order, representing the difference of the population of the nuclear singlet state and the mean population of the three triplets. In many cases, the experiments with long-lived singlet order are very time-consuming because of the need to wait for singlet order decay before the experiment can be repeated; otherwise, spin order remaining from a previous measurement may lead to experimental artifacts. Here, we propose techniques for fast and efficient singlet order destruction. These methods exploit coherent singlet-triplet conversion; in some cases, multiple conversion steps are introduced. We demonstrate that singlet order destruction enables a dramatic reduction of the waiting time between consecutive experiments and suggest to use this approach in singlet-state Nuclear Magnetic Resonance (NMR) experiments with nearly equivalent spins.

11.
J Chem Phys ; 150(6): 064201, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769975

RESUMO

A method is implemented to perform "fast" adiabatic variation of the spin Hamiltonian by imposing the constant adiabaticity condition. The method is applied to improve the performance of singlet-state Nuclear Magnetic Resonance (NMR) experiments, specifically, for efficient generation and readout of the singlet spin order in coupled spin pairs by applying adiabatically ramped RF-fields. Test experiments have been performed on a specially designed molecule having two strongly coupled 13C spins and on selectively isotopically labelled glycerol having two pairs of coupled protons. Optimized RF-ramps show improved performance in comparison, for example, to linear ramps. We expect that the methods described here are useful not only for singlet-state NMR experiments but also for other experiments in magnetic resonance, which utilize adiabatic variation of the spin Hamiltonian.

12.
Phys Chem Chem Phys ; 20(20): 13705-13713, 2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29508877

RESUMO

Long-lived singlet order is exploited in diffusion NMR experiments to successfully measure the tortuosity of randomly packed spheres with diameters ranging from 500 to 1000 µm. The pore spaces in such packings have characteristic length scales well beyond the length scale limit set by spin relaxation in conventional NMR-diffusion experiments. Diffusion times of up to 240 s were used to obtain the restricted diffusion coefficient as a function of diffusion time in the long-time diffusion regime. Experimental results were validated with numerical simulations and data from X-ray micro-computed tomography.

13.
Phys Chem Chem Phys ; 20(15): 9755-9759, 2018 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-29595200

RESUMO

Monodeuterated methyl groups may support a long-lived nuclear spin state, with a relaxation time exceeding the conventional spin-lattice relaxation time T1. Dissolution-DNP (dynamic nuclear polarization) may be used to hyperpolarize such a long-lived spin state. This is demonstrated for the CH2D groups of a piperidine derivative. The polarized sample is manipulated in the ambient magnetic field of the laboratory, without destruction of the hyperpolarized singlet order. Strongly enhanced CH2D signals are observed more than one minute after dissolution, even in the presence of paramagnetic radicals, by which time the NMR signal from the hyperpolarized proton magnetization has completely disappeared.

14.
Proc Natl Acad Sci U S A ; 112(32): 9896-901, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26216996

RESUMO

Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to (15)N-labeled channelrhodopsin-2 carrying 14,15-(13)C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early [Formula: see text] K-like state, the slowly decaying late intermediate [Formula: see text], and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Luz , Espectroscopia de Ressonância Magnética , Rodopsina/metabolismo , Isótopos de Carbono , Domínio Catalítico , Escuridão , Bicamadas Lipídicas/metabolismo , Isótopos de Nitrogênio , Multimerização Proteica , Rodopsina/química
15.
J Labelled Comp Radiopharm ; 61(13): 922-933, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29080288

RESUMO

Three all-trans retinals containing multiple 13 C labels have been synthesized to enable dynamic nuclear polarization enhanced solid-state magic angle spinning NMR studies of novel microbial retinylidene membrane proteins including proteorhodpsin and channelrhodopsin. The synthetic approaches allowed specific introduction of 13 C labels in ring substituents and at different positions in the polyene chain to probe structural features such as ring orientation and interaction of the chromophore with the protein in the ground state and in photointermediates. [10-18-13 C9 ]-All-trans-retinal (1b), [12,15-13 C2 ]-all-trans-retinal (1c), and [14,15-13 C2 ]-all-trans-retinal (1d) were synthesized in in 12, 8, and 7 linear steps from ethyl 2-oxocyclohexanecarboxylate (5) or ß-ionone (4), respectively.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Retinaldeído/química , Retinaldeído/síntese química , Técnicas de Química Sintética , Marcação por Isótopo , Estereoisomerismo
16.
J Am Chem Soc ; 139(45): 16143-16153, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29027800

RESUMO

Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light-induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the deprotonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Bombas de Próton/química , Bombas de Próton/metabolismo , Bombas de Próton/efeitos da radiação , Rodopsinas Microbianas/efeitos da radiação , Bases de Schiff/química
17.
J Org Chem ; 82(17): 8943-8949, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28745884

RESUMO

We have recently shown that the small proton chemical shift difference in 2-methyl-1-(methyl-d)piperidine supports a long-lived nuclear spin state. To identify additional candidate molecules with CH2D groups exhibiting accessible long-lived states, and to investigate the factors governing the magnitude of the shift differences, we report a computational and experimental investigation of methyl rotational equilibria and proton chemical shifts in a variety of 2-substituted 1-(methyl-d)piperidines. The polarity and size of the 2-substituent affect the 1,2-stereoisomeric relationship, and consequently, the strength of the rotational asymmetry within the CH2D group. Nonpolar and large 2-substituents prefer the equatorial position, and relatively large shift differences (i.e., > 13 ppb) are observed. Polar and small substituents, however, increasingly prefer the axial position, and medium to small shift differences (i.e., 0 to 9 ppb) are observed. In addition, the diastereotopic CH2D proton chemical shift difference for tricarbonyl(1-chloro-2-deuteriomethylbenzene) chromium(0) was computed, showing that reasonable predictions of these small shift differences can be extended to more complex, organometallic species.

18.
J Labelled Comp Radiopharm ; 60(2): 135-139, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27990671

RESUMO

Unsymmetrical perdeuterated doubly 13 C labelled oxalates exhibit extended lifetimes in the nuclear singlet state. Synthesis of 1,2 13 C2 oxalates from commercially available precursors is described, facilitating preparation of unsymmetrical oxalates in a controlled manner.


Assuntos
Oxalatos/química , Compostos Radiofarmacêuticos/síntese química , Isótopos de Carbono/química , Compostos Radiofarmacêuticos/química
19.
Phys Chem Chem Phys ; 18(27): 17965-72, 2016 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-27327382

RESUMO

It is possible to access long-lived nuclear singlet order in monodeuterated methyl groups, in the case that a significant chemical shift difference exists between the CH2D protons. This occurs when the local environment is chiral, and the CH2D rotamers have different populations. An experimental demonstration is presented for the case of N-CH2D-2-methylpiperidine. The ratio of the singlet relaxation time constant TS to the longitudinal relaxation time constant T1 is found to be equal to 3.1 ± 0.1, over a wide range of temperatures, solvents, and magnetic fields. The longest observed value of TS approaches 1 minute. The relaxation mechanisms of the long-lived state are discussed, and a modified model of the CH2D geometry is proposed to explain the observed ratio of TS to T1.

20.
Phys Chem Chem Phys ; 17(37): 23748-53, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26299667

RESUMO

Solid-state NMR transitions involving outer energy levels of the spin-1 (14)N nucleus are immune, to first order in perturbation theory, to the broadening caused by the nuclear quadrupole interaction. The corresponding overtone spectra, when acquired in conjunction with magic-angle sample spinning, result in lines, which are just a few kHz wide, permitting the direct detection of nitrogen compounds without the need for labeling. Despite the success of this technique, "overtone" resonances are still broadened due to indirect, second order effects arising from the large quadrupolar interaction. Here we demonstrate that another order of magnitude in spectral resolution may be gained by using double rotation. This brings the width of the (14)N solid-state NMR lines much closer to the region commonly associated with high-resolution solid-state NMR spectroscopy of (15)N and demonstrates the improvements in resolution that may be possible through the development of pulsed methodologies to suppress these second order effects.


Assuntos
Nitrogênio/química , Rotação , Espectroscopia de Ressonância Magnética
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