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Inside of the burst-phase intermediate of a protein folding. Hydration of hydrophobic groups 蛋白质折叠的爆发期中间体内部。疏水基团的水合作用。
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-25 DOI: 10.1016/j.bpc.2024.107268
Elena I. Bolonova , Tatiana N. Melnik , Sergey A. Potekhin

The thermal effect of the formation of the “burst-phase” folding intermediate has been studied using a titration calorimeter. It is shown that, unlike the total thermal effect of native structure formation, it can be both positive and negative depending on the temperature. The reasons for this paradoxical behavior are analyzed. A conclusion is drawn about the leading role of dehydration of non-polar groups in the first stage of folding.

我们使用滴定量热计研究了 "爆发相 "折叠中间体形成的热效应。结果表明,与原生结构形成的总热效应不同,它既可以是正的,也可以是负的,这取决于温度。分析了这种矛盾行为的原因。得出了非极性基团脱水在折叠第一阶段起主导作用的结论。
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引用次数: 0
Ca2+ binding shifts dimeric dual oxidase's truncated EF-hand domain to monomer Ca2+ 结合使二聚体双氧化酶的截短 EF-手结构域转变为单体
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-24 DOI: 10.1016/j.bpc.2024.107271
Chin-Chuan Wei , Amena Abdul Razzak , Hadis Ghasemi , Rahil Khedri , Alexandria Fraase

Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca2+ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca2+ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca2+ binding (Ka = 107 M−1) while EF1 shows a weaker binding (Ka = 105 M−1), resulting in the burial of its negatively charged residues. The Ca2+ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca2+-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca2+ binding is vital for its Duox's domain-domain interaction for electron transfer.

双氧化酶(Duox)产生的过氧化氢对甲状腺激素的合成至关重要。Duox 的活化涉及 Ca2+ 与其 EF 手域(EFD)的结合,EFD 包含两个 EF 手(EFs)。在这项研究中,我们利用光谱法、量热法、电泳迁移率法和凝胶过滤法对截短的 EFD 进行了表征,以获得其 Ca2+ 结合的热力学和动力学,并评估相关的构象变化。我们的研究结果表明,其第 2 EF-手(EF2)与 Ca2+ 的结合放热较强(Ka = 107 M-1),而 EF1 与 Ca2+ 的结合较弱(Ka = 105 M-1),导致其带负电荷的残基被掩埋。Ca2+ 与 EFD 的结合导致其结构稳定,熔化温度从 67°C 上升到 99°C,并诱导其结构从二聚体形式转变为单体形式。EF2似乎在其apo形式的二聚体形成中起作用,而Ca2+结合的EF1的疏水暴露对其holo形式的二聚体形成至关重要。这一结果与低温电子显微镜获得的结构一致,表明 EFD 在与 Ca2+ 结合后具有疏水斑块的稳定结构对其 Duox 结构域-结构域之间的电子传递相互作用至关重要。
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引用次数: 0
The role of anaplerotic metabolism of glucose and glutamine in insulin secretion: A model approach 葡萄糖和谷氨酰胺的非同步代谢在胰岛素分泌中的作用:模型方法
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-23 DOI: 10.1016/j.bpc.2024.107270
Vladimir Grubelnik , Jan Zmazek , Marko Gosak , Marko Marhl

We propose a detailed computational beta cell model that emphasizes the role of anaplerotic metabolism under glucose and glucose-glutamine stimulation. This model goes beyond the traditional focus on mitochondrial oxidative phosphorylation and ATP-sensitive K+ channels, highlighting the predominant generation of ATP from phosphoenolpyruvate in the vicinity of KATP channels. It also underlines the modulatory role of H2O2 as a signaling molecule in the first phase of glucose-stimulated insulin secretion. In the second phase, the model emphasizes the critical role of anaplerotic pathways, activated by glucose stimulation via pyruvate carboxylase and by glutamine via glutamate dehydrogenase. It particularly focuses on the production of NADPH and glutamate as key enhancers of insulin secretion. The predictions of the model are consistent with empirical data, highlighting the complex interplay of metabolic pathways and emphasizing the primary role of glucose and the facilitating role of glutamine in insulin secretion. By delineating these crucial metabolic pathways, the model provides valuable insights into potential therapeutic targets for diabetes.

我们提出了一个详细的β细胞计算模型,强调在葡萄糖和葡萄糖-谷氨酰胺刺激下无机代谢的作用。该模型超越了传统的线粒体氧化磷酸化和 ATP 敏感的 K+ 通道,强调在 KATP 通道附近主要由磷酸烯醇丙酮酸生成 ATP。该模型还强调了 H2O2 作为信号分子在葡萄糖刺激胰岛素分泌第一阶段的调节作用。在第二阶段,该模型强调了葡萄糖刺激通过丙酮酸羧化酶和谷氨酰胺通过谷氨酸脱氢酶激活的无动力通路的关键作用。它特别强调了 NADPH 和谷氨酸的产生是胰岛素分泌的关键促进因素。该模型的预测与经验数据一致,突出了代谢途径之间复杂的相互作用,强调了葡萄糖在胰岛素分泌中的主要作用和谷氨酰胺的促进作用。通过描述这些关键的代谢途径,该模型为糖尿病的潜在治疗目标提供了宝贵的见解。
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引用次数: 0
Characterization of 10MAG/LDAO reverse micelles: Understanding versatility for protein encapsulation 10MAG/LDAO 反向胶束的表征:了解蛋白质封装的多功能性
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-21 DOI: 10.1016/j.bpc.2024.107269
Crystal I. Stackhouse , Kali N. Pierson , Courtney L. Labrecque , Cara Mawson , Joshua Berg , Brian Fuglestad , Nathaniel V. Nucci

Reverse micelles (RMs) are spontaneously organizing nanobubbles composed of an organic solvent, surfactants, and an aqueous phase that can encapsulate biological macromolecules for various biophysical studies. Unlike other RM systems, the 1-decanoyl-rac-glycerol (10MAG) and lauryldimethylamine-N-oxide (LDAO) surfactant system has proven to house proteins with higher stability than other RM mixtures with little sensitivity to the water loading (W0, defined by the ratio of water to surfactant). We investigated this unique property by encapsulating three model proteins – cytochrome c, myoglobin, and flavodoxin – in 10MAG/LDAO RMs and applying a variety of experimental methods to characterize this system's behavior. We found that this surfactant system differs greatly from the traditional, spherical, monodisperse RM population model. 10MAG/LDAO RMs were discovered to be oblate ellipsoids at all conditions, and as W0 was increased, surfactants redistributed to form a greater number of increasingly spherical ellipsoidal particles with pools of more bulk-like water. Proteins distinctively influence the thermodynamics of the mixture, encapsulating at their optimal RM size and driving protein-free RM sizes to scale accordingly. These findings inform the future development of similarly malleable encapsulation systems and build a foundation for application of 10MAG/LDAO RMs to analyze biological and chemical processes under nanoscale confinement.

反向胶束(RM)是一种自发组织的纳米气泡,由有机溶剂、表面活性剂和水相组成,可以封装生物大分子,用于各种生物物理研究。与其他 RM 系统不同,1-癸酰-rac-甘油(10MAG)和月桂基二甲胺-N-氧化物(LDAO)表面活性剂系统已被证明比其他 RM 混合物具有更高的稳定性,而且对水负载(W0,由水与表面活性剂的比例定义)几乎不敏感。我们将细胞色素 c、肌红蛋白和黄独素这三种模型蛋白质封装在 10MAG/LDAO RMs 中,并采用多种实验方法来表征该系统的行为,从而研究了这一独特特性。我们发现,这种表面活性剂系统与传统的球形单分散 RM 群体模型有很大不同。我们发现,10MAG/LDAO RM 在所有条件下都是扁平椭圆体,随着 W0 的增加,表面活性剂重新分布,形成了更多数量越来越多的球形椭圆体颗粒,并汇集了更多类似块状的水。蛋白质对混合物的热力学产生了明显的影响,在其最佳RM尺寸上进行封装,并促使不含蛋白质的RM尺寸相应增大。这些发现为今后开发类似的可塑性封装系统提供了信息,并为应用 10MAG/LDAO RMs 分析纳米尺度约束下的生物和化学过程奠定了基础。
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引用次数: 0
Critical assessment of popular biomolecular force fields for molecular dynamics simulations of folding and enzymatic activity of main protease of coronavirus SARS-CoV-2 对用于冠状病毒 SARS-CoV-2 主要蛋白酶折叠和酶活性分子动力学模拟的常用生物分子力场进行严格评估
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-16 DOI: 10.1016/j.bpc.2024.107258
Kateryna O. Lohachova, Alexander Kyrychenko, Oleg N. Kalugin

The main cysteine protease (Mpro) of coronavirus SARS-CoV-2 has become a promising target for computational development in anti-COVID-19 treatments. Here, we benchmarked the performance of six biomolecular molecular dynamics (MD) force fields (OPLS-AA, CHARMM27, CHARMM36, AMBER03, AMBER14SB and GROMOS G54A7) and three water models (TIP3P, TIP4P and SPC) for reproducing the native fold and the enzymatic activity of Mpro as monomeric and dimeric units. The MD sampling up to 1 μs suggested that the proper choice of the force fields and water models plays an essential role in reproducing the tertiary structure and the inter-residue distance between the catalytic dyad His41-Cys145. We found that while most benchmarked all-atom force fields reproduce well the native fold of Mpro, the CHARMM27/TIP3P and OPLS-AA/TIP4P setups revealed a good performance in reproducing the structure of the catalytic domain. In addition, these FF setups were also well-adopted for MD sampling of Mpro at the physiologic conditions by mimicking the presence of 100 mM NaCl and the elevated temperature of 310 K. Finally, both FFs were also performed well in reproducing the native fold of Mpro in a dimeric form. Therefore, comparing the preservation of the native fold of Mpro and the stability of its catalytic site architecture, our MD benchmarking suggests that the OPLS-AA/TIP4P and CHARMM27/TIP3P MD setups at the physiologic conditions may be well-suited for rapid in silico screening and developing broad-spectrum anti-coronaviral therapeutic agents.

冠状病毒SARS-CoV-2的主要半胱氨酸蛋白酶(Mpro)已成为抗COVID-19治疗的一个有希望的计算开发目标。在此,我们对六个生物分子分子动力学(MD)力场(OPLS-AA、CHARMM27、CHARMM36、AMBER03、AMBER14SB 和 GROMOS G54A7)和三个水模型(TIP3P、TIP4P 和 SPC)的性能进行了基准测试,以再现 Mpro 作为单体和二聚体单元的原生折叠和酶活性。高达 1 μs 的 MD 采样表明,力场和水模型的正确选择在重现三级结构和催化二元 His41-Cys145 之间的残基间距方面起着至关重要的作用。我们发现,虽然大多数基准全原子力场都能很好地再现 Mpro 的原生折叠,但 CHARMM27/TIP3P 和 OPLS-AA/TIP4P 设置在再现催化结构域的结构方面表现良好。此外,通过模拟 100 mM NaCl 的存在和 310 K 的高温,这些 FF 设置也非常适合在生理条件下对 Mpro 进行 MD 采样。因此,通过比较 Mpro 的原生折叠及其催化位点结构的稳定性,我们的 MD 基准测试表明,生理条件下的 OPLS-AA/TIP4P 和 CHARMM27/TIP3P MD 设置可能非常适合于快速硅学筛选和开发广谱抗oronaviral 治疗药物。
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引用次数: 0
Unveiling the characteristics of D4 and R4 aptamers for their future use in prostate cancer clinical practice 揭示D4和R4适配体的特性,促进其在前列腺癌临床实践中的应用
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-14 DOI: 10.1016/j.bpc.2024.107259
Esther Campos-Fernández, Nathalia Oliveira Alqualo, Emília Rezende Vaz, Cláudia Mendonça Rodrigues, Vivian Alonso-Goulart

The DNA and RNA aptamers D4 and R4, respectively, emerged from the modification of PC-3 cell-binding aptamer A4. Our objective was to characterize the aptamers in silico and in vitro and begin to identify their target molecules. We represented their structures using computational algorithms; evaluated their binding to several prostate cell lines and their effects on the viability and migration of these cells; and determined their dissociation constant by flow cytometry. We analyzed circulating prostate tumor cells from patients using D4, R4, anti-CD133 and anti-CD44. Finally, the target proteins of both aptamers were precipitated and identified by mass spectrometry to simulate their in silico docking. The aptamers presented similar structures and bound to prostate tumor cells without modifying the cellular parameters studied, but with different affinities. The ligand cells for both aptamers were CD44+, indicating that they could identify cells in the mesenchymal stage of the metastatic process. The possible target proteins NXPE1, ADAM30, and MUC6 need to be further studied to better understand their interaction with the aptamers. These results support the development of new assays to determine the clinical applications of D4 and R4 aptamers in prostate cancer.

DNA和RNA适配体D4和R4分别产生于对PC-3细胞结合适配体A4的修饰。我们的目标是在硅学和体外鉴定这些适配体,并开始确定它们的靶分子。我们用计算算法表示了它们的结构;评估了它们与几种前列腺细胞系的结合及其对这些细胞的活力和迁移的影响;并通过流式细胞仪测定了它们的解离常数。我们使用 D4、R4、抗 CD133 和抗 CD44 分析了患者的循环前列腺肿瘤细胞。最后,我们沉淀了这两种适配体的靶蛋白,并通过质谱鉴定来模拟它们的硅对接。这两种适配体结构相似,都能与前列腺肿瘤细胞结合,不会改变所研究的细胞参数,但亲和力不同。两种适配体的配体细胞都是 CD44+,表明它们可以识别转移过程中处于间质阶段的细胞。可能的靶蛋白 NXPE1、ADAM30 和 MUC6 还需要进一步研究,以更好地了解它们与适配体的相互作用。这些结果支持开发新的检测方法,以确定 D4 和 R4 合体在前列腺癌中的临床应用。
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引用次数: 0
Investigating the role of axonal ion channel cooperativity in action potential dynamics: Studies on Hodgkin-Huxley's model 研究轴突离子通道合作性在动作电位动力学中的作用:霍奇金-赫胥黎模型研究
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-08 DOI: 10.1016/j.bpc.2024.107257
Jitender Kumar , Patrick Das Gupta , Subhendu Ghosh

Voltage-gated ion channels play an important role in generating action potential in neurons. These ion channels are found to be in localized cluster form on the axonal membrane surface and behave cooperatively. However, in Hodgkin & Huxley's model of action potential the ion channels are considered to function independently. According to some recent reports, the activity of an ion channel is influenced by the neighboring ion channels' activities. We have modified the Hodgkin-Huxley's model based on our previous studies on cooperativity among ion channels. Computational analysis of the proposed model shows that the initiation of the action potential, amplitude and hyperpolarization are affected significantly by the cooperative interactions among the voltage-gated ion channels present on the axonal membrane surface. These results are qualitatively supported by the existing experimental facts.

电压门控离子通道在神经元产生动作电位的过程中发挥着重要作用。人们发现这些离子通道在轴突膜表面以局部集群的形式存在,并且相互配合。然而,在霍奇金与赫胥黎的动作电位模型中,离子通道被认为是独立发挥作用的。根据最近的一些报道,一个离子通道的活性受相邻离子通道活性的影响。我们根据之前对离子通道间合作性的研究,对霍奇金-赫胥黎模型进行了修改。对所提出模型的计算分析表明,轴突膜表面的电压门控离子通道之间的合作性相互作用对动作电位的启动、振幅和超极化有显著影响。这些结果得到了现有实验事实的定性支持。
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引用次数: 0
Improved analysis of NMR chemical shift perturbations through an error estimation method 通过误差估算方法改进核磁共振化学位移扰动分析
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-06 DOI: 10.1016/j.bpc.2024.107255
Kyoko Furuita , Chojiro Kojima

In solution NMR, chemical shift perturbation (CSP) experiments are widely employed to study intermolecular interactions. However, excluding the nonsignificant peak shift is difficult because little is known about errors in CSP. Here, to address this issue, we introduce a method for estimating errors in CSP based on the noise level. First, we developed a technique that involves line shape fitting to estimate errors in peak position via Monte Carlo simulations. Second, this technique was applied to estimate errors in CSP. In intermolecular interaction analysis of VAP-A with SNX2, error estimation of CSP enabled the evaluation of small but significant changes in peak position and yielded detailed insights that are unattainable with conventional CSP analysis. Third, this technique was successfully applied to estimate errors in residual dipolar couplings. In conclusion, our error estimation method improves CSP analysis by excluding the nonsignificant peak shift.

在溶液核磁共振中,化学位移扰动(CSP)实验被广泛用于研究分子间相互作用。然而,由于对 CSP 中的误差知之甚少,排除不重要的峰移十分困难。为了解决这个问题,我们在此介绍一种基于噪声水平估算 CSP 误差的方法。首先,我们开发了一种涉及线形拟合的技术,通过蒙特卡罗模拟来估计峰值位置的误差。其次,将该技术应用于估计 CSP 中的误差。在 VAP-A 与 SNX2 的分子间相互作用分析中,通过估计 CSP 误差,可以评估峰位微小但显著的变化,并获得传统 CSP 分析无法获得的详细见解。第三,这项技术成功地应用于估计残余偶极耦合的误差。总之,我们的误差估计方法排除了不重要的峰值偏移,从而改进了 CSP 分析。
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引用次数: 0
Exploring the physicochemical properties of the integration of Tristearoyl uridine in Langmuir monolayers: An approach to cell membrane modeling for prodrugs 探索三苯甲酰基尿苷在朗缪尔单层中整合的物理化学特性:原药细胞膜建模方法
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-06 DOI: 10.1016/j.bpc.2024.107256
Felipe Almeida Moreira , Jhon Fernando Berrío Escobar , Cristiano Giordani , Luciano Caseli

Understanding the mechanisms by which drugs interact with cell membranes is crucial for unraveling the underlying biochemical and biophysical processes that occur on the surface of these membranes. Our research focused on studying the interaction between an ester-type derivative of tristearoyl uridine and model cell membranes composed of lipid monolayers at the air-water interface. For that, we selected a specific lipid to simulate nontumorigenic cell membranes, namely 1,2-dihexadecanoyl-sn-glycero-3-phospho-l-serine. We noted significant changes in the surface pressure-area isotherms, with a noticeable shift towards larger areas, which was lower than expected for ideal mixtures, indicating monolayer condensation. Furthermore, the viscoelastic properties of the interfacial film demonstrated an increase in both the elastic and viscous parameters for the mixed film. We also observed structural alterations using vibrational spectroscopy, which revealed an increase in the all-trans to gauche conformers ratio. This confirmed the stiffening effect of the prodrug on the lipid monolayer. In summary, this study indicates that this lipophilic prodrug significantly impacts the lipid monolayer's thermodynamic, rheological, electrical, and molecular characteristics. This information is crucial for understanding how the drug interacts with specific sites on the cellular membrane. It also has implications for drug delivery, as the drug's passage into the cytosol may involve traversing the lipid bilayer.

了解药物与细胞膜相互作用的机制对于揭示发生在这些膜表面的潜在生物化学和生物物理过程至关重要。我们的研究重点是研究三苯甲酰基尿苷的酯类衍生物与由脂质单层组成的模型细胞膜在空气-水界面上的相互作用。为此,我们选择了一种特定的脂质来模拟非致癌细胞膜,即 1,2-二十六碳酰-sn-甘油-3-磷-l-丝氨酸。我们注意到表面压力-面积等温线发生了重大变化,明显向更大面积转变,低于理想混合物的预期,表明存在单层凝结。此外,界面薄膜的粘弹特性表明,混合薄膜的弹性和粘性参数都有所增加。我们还利用振动光谱观察到了结构的变化,发现全反式与高顺式构象比有所增加。这证实了原药对脂质单层的增硬作用。总之,这项研究表明,这种亲脂原药对脂质单分子层的热力学、流变学、电学和分子特性有显著影响。这些信息对于了解药物如何与细胞膜上的特定位点相互作用至关重要。由于药物进入细胞膜可能需要穿越脂质双分子层,因此这对药物输送也有影响。
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引用次数: 0
High-resolution heteronuclear correlations between spin-1/2 and half-integer quadrupolar nuclei under fast MAS solid-state NMR 快速 MAS 固态 NMR 下自旋-1/2 和半整数四极核之间的高分辨率异核相关性
IF 3.8 3区 生物学 Q2 Biochemistry, Genetics and Molecular Biology Pub Date : 2024-05-03 DOI: 10.1016/j.bpc.2024.107254
Manoj Kumar Pandey , Yusuke Nishiyama

High isotropic resolution is essential for the structural elucidation of samples with multiple sites. In this study, utilizing the benefits of TRAPDOR-based heteronuclear multiple quantum coherence (T-HMQC) and a pair of one rotor period long cosine amplitude modulated low-power (cos-lp) pulse-based symmetric-split-t1 multiple-quantum magic angle spinning (MQMAS) methods, we have developed a proton-detected 2D 35Cl/1H T-HMQC-MQMAS pulse sequence under fast MAS (70 kHz) to achieve high-resolution in the indirect dimension of the spin-3/2 (35Cl) nuclei connected via protons. As T-HMQC polarizes not only single-quantum central transition (SQCT) but also triple-quantum (TQ) coherences, the proposed 2D pulse sequence is implemented via selection of two coherence pathways (SQCT TQ SQCT and TQ SQCT TQ) resulting in the 35Cl isotropic dimension and is superior to the existing double-quantum satellite-transition (DQST) T-HMQC in terms of resolution.

高各向同性分辨率对于阐明具有多个位点的样品结构至关重要。在本研究中,我们利用基于 TRAPDOR 的异核多重量子相干(T-HMQC)和一对基于对称-分裂-t1 多量子魔角旋转(MQMAS)的长余弦振幅调制低功率(cos-lp)脉冲的优势、我们在快速 MAS(70 kHz)条件下开发了质子探测二维 35Cl/1H T-HMQC-MQMAS 脉冲序列,以实现通过质子连接的自旋-3/2(35Cl)原子核间接维度的高分辨率。由于 T-HMQC 不仅能极化单量子中心转变(SQCT),还能极化三量子(TQ)相干,因此建议的二维脉冲序列是通过选择两种相干途径(SQCT →TQ →SQCT 和 TQ → SQCT →TQ)来实现的,从而得到 35Cl 的各向同性维度,在分辨率方面优于现有的双量子卫星转变(DQST)T-HMQC。
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引用次数: 0
期刊
Biophysical chemistry
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