NMR技术在蛋白质-蛋白质相互作用研究中的应用——泛素-蛋白水解酶体通路研究实例介绍

蛋白质-蛋白质相互作用在多种细胞内生理活动中发挥关键性作用,而蛋白质复合物结构信息的获得主要依赖于X-射线衍射技术和核磁共振技术2种主要技术手段的使用. 需要指出的是,虽然大部分蛋白质复合物的结构解析使用了X-射线衍射技术,然而在包括无法获得蛋白质复合物晶体、 蛋白质与蛋白质结合强度较弱以及蛋白质复合物系统具有复杂的动力学行为等几种情况下,核磁共振技术是可用于蛋白质复合物结构测定的唯一手段. 用于蛋白质-蛋白质相互作用研究的NMR技术主要有化学位移扰动分析、分子间NOE的检测、顺磁弛豫增强技术、残余偶极耦合检测技术等几种. 该文将结合这几种技术在泛素-蛋白水解酶体通路领域的应用实例对它们的工作原理以及可提供的信息做出总结介绍.     

Improvement of resolution in solid state NMR spectra with J-decoupling: an analysis of lineshape contributions in uniformly C-enriched amino acids and proteins

In magic angle spinning (MAS) NMR spectra of highly and uniformly 13C,15N-enriched amino acids and proteins, homo-nuclear coupling interactions contribute significantly to the 13C linewidths, particularly for moderate applied magnetic field strengths and sample spinning frequencies. In this work, we attempted to dissect, analyze, and control the contributions of J-coupling and residual homo-nuclear dipolar coupling interactions to the linewidths of uniformly 13C,15N-enriched crystalline alanine; these studies were carried out at 9.4 T using a range of spinning frequencies from 5 to 15 kHz. The anisotropic second-order dipolar shifts and the J-splittings are comparable in their contribution to the linewidths, but behave very differently in terms of experimental protocols for line narrowing. In contrast to the J-coupling interactions, the second-order dipolar broadening cannot be refocused using selective pulses on the passively coupled spin. We carried out experiments to remove or refocus the 13C J-coupling interactions (omega1 J-decoupling) using a selective DANTE pulse in the center of the indirect evolution period. Inversion profiles and bandwidths of selective DANTE pulses acting on transverse magnetization, in the regime of moderate spinning frequencies, were characterized computationally and experimentally. A dramatic improvement in the resolution of the 2D spectrum was achieved when this decoupling protocol was employed.     

Side-chain H and C resonance assignment in protonated/partially deuterated proteins using an improved 3D(13)C-detected HCC-TOCSY

We propose the use of (13)C-detected 3D HCC-TOCSY experiments for assignment of (1)H and (13)C resonances in protonated and partially deuterated proteins. The experiments extend 2D C-13-start and C-13-observe TOCSY type experiments proposed earlier. Introduction of the third (1)H dimension to 2D TOCSY: (i) reduces the peak overlap and (ii) increases the sensitivity per unit time, even for highly deuterated (>85%) protein samples, which makes this improved method an attractive tool for the side-chain H and C assignment of average sized proteins with natural isotope abundance as well as large partially deuterated proteins. The experiments are demonstrated with a 16 kDa (15)N, (13)C-labeled non-deuterated apo-CcmE and a 48 kDa uniformly (15)N, (13)C-labeled and fractionally ( approximately 90%) deuterated dimeric sFkpA. It is predicted that this method should be suitable for the assignment of methyl (13)C and (1)H chemical shifts of methyl protonated, highly deuterated and (13)C-labeled proteins with even higher molecular weight.     

13C-NMR Assignments for Methyls and Quaternary Carbons of the Limonoid Obacunone

The high field ¹H and ¹³C resonances of obacunone were established by a series of I D and 2D NMR experiments. The ¹³C-NMR signals of the five tertiary methyl groups and all quatenary carbons were unambiguously assigned. The results confirm the stereochemical assignments at all stereogenic centres.     

A new detection scheme for ultrafast 2D J-resolved spectroscopy

Recent ultrafast techniques enable 2D NMR spectra to be obtained in a single scan. A modification of the detection scheme involved in this technique is proposed, permitting the achievement of 2D 1H J-resolved spectra in 500 ms. The detection gradient echoes are substituted by spin echoes to obtain spectra where the coupling constants are encoded along the direct nu2 domain. The use of this new J-resolved detection block after continuous phase-encoding excitation schemes is discussed in terms of resolution and sensitivity. J-resolved spectra obtained on cinnamic acid and 3-ethyl bromopropionate are presented, revealing the expected 2D J-patterns with coupling constants as small as 2 Hz.     

A Definitive Example of Aluminum-27 Chemical Shielding Anisotropy

Solid-state²⁷Al NMR spectra have been obtained for a crystalline 1:1 complex of AlCl₃and OPCl₃. Aluminum chloride phosphoryl chloride, AlCl₃· OPCl₃(1), is unusual in that the Al–O–P bond angle is close to 180°. From analysis of the²⁷Al MAS NMR spectra, it was determined that the²⁷Al nuclear quadrupole coupling constant is 6.0(1) MHz, the asymmetry in the electric field gradient (efg) tensor is 0.15(2), and the isotropic chemical shift, δ_iso(²⁷Al), is 88(1) ppm. Solid-state²⁷Al NMR of a stationary sample reveals a line shape affected by a combination of anisotropic chemical shielding and second-order quadrupolar interactions. Analysis of this spectrum yields a chemical shift anisotropy of 60(1) ppm and orientations of the chemical shift and electric field gradient tensors in the molecular frame. Experimental results are compared with those calculated usingab initioHartree–Fock and density functional theory.     

Resolution and sensitivity aspects of ultrafast J-resolved 2D NMR spectra

Recent ultrafast techniques enable nD NMR spectra to be obtained in a single scan. However, resolution enhancement in the ultrafast domain leads to important sensitivity losses and lineshape distortions. In order to understand better resolution and spatial encoding aspects of continuous phase-encoding schemes, a theoretical and experimental comparison of different excitation patterns is carried out. Molecular diffusion appears to be the main cause of signal-to-noise ratio decrease, and a multi-echo excitation scheme is proposed to limit its effects when a good resolution is needed. Results obtained on 2D J-resolved spectra are presented.     

Assignment of the 1H and 13C Spectra of 5-Methylthio-4H-1-(p-R1-phenyl)-3a-(p-R2-phenyl)-3a,4-dihydro (1,2,4) oxadiazolo(4,5-a), 1,5-benzodiazepines using 2D NMR Spectroscopy.

The ¹H NMR spectra of the title benzodiazepines derivatives is highly congested because all the protons are in aromatic enviroment so many proton signals remain overlap even 300 MHz or higher fields. With this in mind, the assignment of the ¹H and ¹³C spectra of these compounds obtained using COSY, NOESY, HMQC and HMBC experiments is reported.     

Assignment of the 1H and 13C Spectra of 5-Methylthio-4H-1-(p-R1-phenyl)-3a-(p-R2-Phenyl)-3a, 4-dihydro (1,2,4) oxadiazolo(4,5-a), 1, 5-benzodiazepines Using 2D NMR Spectroscopy

The ¹H NMR spectra of the title benzodiazepines derivatives is highly congested because all the protons are in aromatic environment so many proton signals remain overlap even 300 MHz or higher fields. With this in mind, the assignment of the ¹H and ¹³C spectra of these compounds obtained using COSY, NOESY, HMQC and HMBC experiments is reported.     

Biomagnetic measurements utilising a superparamagnetic marker: a feasibility study

Summary A preliminary study is here reported on a new potential marker for biomagnetic measurements. The marker consists of superparamagnetic polymer microspheres which were detected in the presence of external steady magnetic fields by means of an r.f.-SQUID magnetometer. The particles were prepared in samples differing in the concentration value and immersed in a homogeneous magnetic field of variable intensity. A simple model was taken into account for the distribution of the microspheres in the samples, so that the theoretical values were compared to the marker field values measured by the biomagnetic sensor. The overall sensitivity of the experimental apparatus and the minimum concentration value of the marker were then estimated.     

A biomagnetic method for studying gastro-intestinal activity

Summary A novel method to investigate the gastro-intestinal activity by means of a superconducting instrumentation based on SQUID sensors developed for biomagnetic measurements is presented. The magnetic field generated by a small marker ingested by the subject is measured over the abdomen. The localization of the marker,i.e. a magnetic dipole, is carried out by means of a standard algorithm used in biomagnetic studies. In this way it is possible to noninvasively study the activity of the gastro-intestinal system. This new approach may lead to important clinical applications.     

Accurate Measurement of Small Spin–Spin Couplings in Partially Aligned Molecules Using a Novel J-Mismatch Compensated Spin-State-Selection Filter

The accurate measurement of small spin–spin coupling constants in macromolecules dissolved in a liquid crystalline phase is important in the context of molecular structure investigation by modern liquid state NMR. A new spin-state-selection filter, DIPSAP, is presented with significantly reduced sensitivity to J-mismatch of the filter delays compared to previously proposed pulse sequences. DIPSAP presents an attractive new approach for the accurate measurement of small spin–spin coupling constants in molecules dissolved in anisotropic solution. Application to the measurement of ¹⁵N–¹³C′ and ¹H^N–¹³C′ coupling constants in the peptide planes of ¹³C, ¹⁵N labeled proteins demonstrates the high accuracy obtained by a DIPSAP-based experiment.     

Covariant tensor formalism for partial-wave analyses of ψ decays into γB¯, γγV and ψ(2s)↦γχ<Subscript>c0,1,2</Subscript> with χ<Subscript>c0,1,2</Subscript>↦K¯πand 2π<Superscript>+</Superscript>2π<Superscript>-</Superscript>

With accumulation of high statistics data at BES and CLEO-c, many new interesting channels can get enough statistics for partial-wave analysis (PWA). Among them, ψ↦γpˉ,γΛˉ,γΣˉ,γΞˉ channels provide a good place for studying baryon-antibaryon interactions; the double radiative decays ψ↦γγV with V ≡ ρ,ω,φ have a potential to provide information on the flavor content of any meson resonances (R) with positive charge parity (C = +) and mass above 1 GeV through ψ↦γR↦γγV; ψ(2s)↦γχ_c0,1,2 with χ_c0,1,2↦Kˉπ⁺π^- and 2π⁺2π^- decays are good processes to study χ_cJ charmonium decays. Using the covariant tensor formalism, here we provide theoretical PWA formulae for these channels.     

利用EPR研究三氧化钼中氧还活性位点形成的温度依赖性

本文利用热处理还原法,利用三氧化钼诱导形成具有氧化还原活性的中间产物及其温度相关特性. 电子顺磁共振波谱(EPR)实验结果表明,热处理后在Mo^VI离子被还原成Mo^V离子并形成了超氧阴离子自由基O₂^-. 这两种活性中心的最佳热还原温度约300∽350 ^°C. 自350 ^°逐渐升温,两种EPR信号强度迅速下降,下降∽50%所对应的温度是400∽450 ^°C;当温度升至600 ^°C或更高时,EPR信号降至0. 结果表明,较高温处理或持续长时间处理都使热还原更容易发生,积累了越来越多的电子,在基底中形成类似于反铁磁耦合的电子离域. 随着离域程度增大,EPR信号强度开始下降并直至消失.     

Measurement of Small One-Bond Proton–Carbon Residual Dipolar Coupling Constants in Partially Oriented C Natural Abundance Oligosaccharide Samples: Analysis of Heteronuclear JCH-Modulated Spectra with the BIRD Inversion Pulse

Two 2D J-modulated HSQC-based experiments were designed for precise determination of small residual dipolar one-bond carbon–proton coupling constants in ¹³C natural abundance carbohydrates. Crucial to the precision of a few hundredths of Hz achieved by these methods was the use of long modulation intervals and BIRD pulses, which acted as semiselective inversion pulses. The BIRD pulses eliminated effective evolution of all but ¹J_CH couplings, resulting in signal modulation that can be described by simple modulation functions. A thorough analysis of such modulation functions for a typical four-spin carbohydrate spin system was performed for both experiments. The results showed that the evolution of the ¹H–¹H and long-range ¹H–¹³C couplings during the BIRD pulses did not necessitate the introduction of more complicated modulation functions. The effects of pulse imperfections were also inspected. While weakly coupled spin systems can be analyzed by simple fitting of cross peak intensities, in strongly coupled spin systems the evolution of the density matrix needs to be considered in order to analyse data accurately. However, if strong coupling effects are modest the errors in coupling constants determined by the “weak coupling” analysis are of similar magnitudes in oriented and isotropic samples and are partially cancelled during dipolar coupling calculation. Simple criteria have been established as to when the strong coupling treatment needs to be invoked.     

Application of the Filter Diagonalization Method to One- and Two-Dimensional NMR Spectra

A new non-Fourier data processing algorithm, the filter diagonalization method (FDM), is presented and applied to phase-sensitive 1D and 2D NMR spectra. FDM extracts parameters (peak positions, linewidths, amplitudes, and phases) directly from the time-domain data by fitting the data to a sum of damped complex sinusoids. Grounded in a quantum-mechanical formalism, FDM shares some of the features of linear prediction and other linear algebraic approaches, but is numerically more efficient, scaling like the fast Fourier transform algorithm with respect to data size, and has the ability to correctly handle spectra with thousands or even millions of lines where the competing methods break down. Results obtained on complex spectra are promising.     

Breakup of 8B and the 7Be(p, γ)8B reaction

The calculated rate of events in some of the existing solar neutrino detectors is directly proportional to the rate of the ⁷Be(p, γ)⁸B reaction measured in the laboratory at low energies. However, the low-energy cross sections of this reaction are quite uncertain as various measurements differ from each other by 30–40%. The Coulomb dissociation process which reverses the radiative capture by the dissociation of ⁸B in the Coulomb field of a target, provides an alternate way of accessing this reaction. While this method has several advantages (like large breakup cross sections and flexibility in the kinematics), the difficulties arise from the possible interference by the nuclear interactions, uncertainties in the contributions of the various multipoles and the higher order effects, which should be considered carefully. We review the progress made so far in the experimental measurements and theoretical analysis of the breakup of ⁸B and discuss the current status of the low-energy cross sections (or the astrophysical S-factor) of the ⁷Be(p, γ)⁸B reaction extracted therefrom. The future directions of the experimental and theoretical investigations are also suggested. Work supported by EPSRC, UK, grant nos J/95867 and L/94574.