REVIEW: High pressure NMR study of proteins – seeking roots for function,evolution, disease and food applications

NMR experiments at variable pressure reveal a wide range of conformation of a globular protein spanning from within the folded ensemble to the fully unfolded ensemble, herewith collectively called “high-energy conformers”. The observation of “high-energy conformers” in a wide variety of globular proteins has led to the “volume theorem”: the partial molar volume of a protein decreases with the decrease in its conformational order. Since “high-energy conformers” are intrinsically more reactive than the basic folded conformer, they could play decisive roles in all phenomena of proteins, namely function, environmental adaptation and misfolding. Based on the information on high-energy conformers and the rules on their partial volume in its monomeric state and amyloidosis, one may have a general view on what is happening on proteins under pressure. Moreover, one may even choose a high-energy conformer of a protein with pressure as variable for a particular purpose. Bridging “high-energy conformers” to macroscopic pressure effects could be a key to success in pressure application to biology, medicine, food technology and industry in the near future.     

Nuclear magnetic relaxation and dynamics of biopolymers

A model is proposed in which the NMR relaxation dispersion profiles of biomolecule water solutions are interpreted in terms of magnetic field fluctuations due to the backbone vibrations involving the biopolymer paramagnetic nuclei. This model, which takes into account the experimental evidence which has appeared in the literature for a “fractal-like” density of vibrational state for the protein backbone, has been used to fit some experimental NMR relaxation profiles of biomolecule water solutions. The results obtained are discussed critically in connection with the data reported in the related literature.     

Cooling overall spin temperature: protein NMR experiments optimized for longitudinal relaxation effects

In experiments performed on protonated proteins at high fields, 80% of the NMR spectrometer time is spent waiting for the ¹H atoms to recover their polarization after recording the free induction decay. Selective excitation of a fraction of the protons in a large molecule has previously been shown to lead to faster longitudinal relaxation for the selected protons [K. Pervushin, B. Vögeli, A. Eletsky, Longitudinal ¹H relaxation optimization in TROSY NMR spectroscopy, J. Am. Chem. Soc. 124 (2002) 12898–12902; P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015; H.S. Attreya, T. Szyperski, G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment, Proc. Natl. Acad. Sci. USA 101 (2004) 9642–9647]. The pool of non-selected protons acts as a “thermal bath” and spin-diffusion processes (“flip-flop” transitions) channel the excess energy from the excited pool to the non-selected protons in regions of the molecule where other relaxation processes can dissipate the excess energy. We present here a sensitivity enhanced HSQC sequence (COST-HSQC), based on one selective E-BURP pulse, which can be used on protonated ¹⁵N enriched proteins (with or without ¹³C isotopic enrichment). This experiment is compared to a gradient sensitivity enhanced HSQC with a water flip-back pulse (the water flip-back pulse quenches the spin diffusion between ¹H^N and ¹H^α spins). This experiment is shown to have significant advantages in some circumstances. Some observed limitations, namely sample overheating with short recovery delays and complex longitudinal relaxation behaviour are discussed and analysed.     

生物分子结合水的结构与动力学研究进展

生物结合水在维护生物大分子的结构、稳定性以及调控动力学性质和生理功能等方面起着决定性的作用.从分子水平上理解生物结合水分子的结构与性质及其影响生物结构和功能的本质与规律,是揭示生物大分子生理功能机理的关键.目前生物结合水的结构与动力学相关研究尚处于初步阶段.本文从三个方面介绍当前生物结合水的相关研究及其进展:首先介绍结合水对蛋白质折叠、质子给予与迁移、配体结合与药物设计以及变构效应等生物结构和功能的影响;然后介绍生物分子周围的水分子结构研究情况;最后从时间尺度、动力学属性、生物分子与水分子之间的动力学耦合作用、蛋白质表面结合水次扩散运动等角度介绍生物分子水合动力学的研究进展,并归纳出一些目前尚待进一步解决的科学问题.     

采用FTIR技术研究高压处理对冻干大豆分离蛋白构象的影响

采用傅里叶转换红外光谱技术(FTIR)研究了高压(HP)处理对冷冻干燥的大豆分离蛋白(SPI)构象的影响。在SPI的去卷积FTIR光谱的酰胺Ⅰ′区域(1 600～1 700 cm-1),观察到12个与蛋白构象相关的红外吸收峰,分别对应于CO键伸缩振动与肽键的C—N伸缩振动。通过对该区域的峰强度与波数分析显示,压力为200～400 MPa的HP处理导致SPI在该区域的峰发生明显的“红移”(约2 cm-1),强度也显著增加。更高的HP处理进一步增强了SPI的酰胺Ⅰ′区域的峰强度。对酰胺Ⅱ峰分析显示,HP处理导致酰胺Ⅱ峰(如1 560～1 500 cm-1)的强度、面积逐渐增加(与压力呈正相关)。以上分析显示,HP处理导致SPI的二级与三级结构逐渐打开,然而变性蛋白的结构在高压释放后经历一个“重构过程”。     

Radical reactions in organic crystals under pressure: Experiment and theory

The kinetics of radical transformations in gamma-irradiated dicarboxylic acid single crystals at a high pressure was studied by EPR spectroscopy. The activation parameters of the detachment of the hydrogen and reaction coordinates were determined. The dependence of the rate constant for the reaction on the pressure and crystal physical parameters was studied. The compression of crystals decelerated the detachment of hydrogen (the activation volumes were positive), and the rate-determining stage of the process was free volume fluctuations responsible for the approach of reagent active centers to each other and their arrangement and orientation along the reaction coordinate on the potential energy surface. The kinetic parameters of the reaction are closely related to the crystal characteristics such as the molar volume, compressibility, and thermal expansion coefficient. A theory describing this relation is suggested. Only part of the free volume was shown to be used for “reaction” fluctuations. The types of reagent motions responsible for the molecular organization of the transition state were identified.     

Suppression of inhomogeneous line broadening of Mössbauer resonance by a spinning magnetic field

A spinning magnetic field, satisfying the so-called “magic-angle” condition, is widely used for the suppression of inhomogeneous line broadening of spin transitions in nuclear magnetic resonance (NMR) spectroscopy. We show that the redefined “magic-angle” condition can also be used for the narrowing of γ-ray nuclear transitions in the Mössbauer spectroscopy.     

Rietveld analysis of high pressure powder diffraction data

Abstract

X-ray and neutron powder diffraction data obtained from samples contained within high pressure cells are generally of lower quality than data collected from samples at ambient conditions. The far smaller sample size as well as possible contamination of the pattern by the pressure cell means that Rietveld refinement techniques must be adapted to extract the maximum useful information from the data. These problems become paramount as larger structures at high pressure are attempted. Techniques such as “leBail extraction”, “soft restraints” and “rigid body refinement” will be discussed with application to analysis of high pressure neutron powder diffraction data.     

Phase transitions of sulfur at high pressure: Influence of temperature and pressure environment

Abstract

Phase transitions of orthorhombic sulfur were investigated above 10 GPa by Raman spectroscopy using red light excitation. Transitions into several phases that have been reported in previous studies using green light excitation, are confirmed. The phase behaviour is observed to depend strongly on the preparation method. In the presence of a pressure transmitting medium (methanol/ethanol, 4:1), a sequence of phases α-S₈ → [intermediate phase (“ip”) + S₆] → [S₆ + high pressure-low temperature phase (“hplt”)] is described and characterized. Without the use of a pressure transmitting medium, the phase sequence α-S₈ → [“ip” + “hplt”] + “hplt” is observed. In addition, contributions of amorphous sulfur are detected around 10 GPa, i.e. at pressures below the transformation of α-S₈ into the above-mentioned phases. Characteristic Raman spectra of the different phases are extracted and documented over a wide pressure range.     

Mixing apparatus for preparing NMR samples under pressure

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.     

Potential applications of high pressures in pharmaceutical science and medicine

Abstract

High pressure (HP) technology appears to be of paramount interest for valuable applications in pharmacy and medicine, owing to academic studies and industrial developments of this technology for food processing. The main potential application of HP between 100 MPa and 1200 MPa (1 kbar to 12kbar) is the inactivation of biological agents (bacteria, moulds, yeasts, virus and even prions) which allows the sterilization (i.e, “pascalization”) of thermo-, chemo-, or radio-sensitive materials and chemicals. The immunogenicity of some pressure-killed bacteria or virus suggests also the potentiality of making new vaccines. Another interest of the HP technology is the possibility of storage at subzero temperatures without freezing of cells and animal tissues to be used for cells and organs for transplant. HP engineering may be used for enzyme-catalyzed synthesis of fine chemicals, production of modified proteins of medical or pharmaceutical interest, and organic synthesis of pharmaceuticals.     

Probing structural integrity of single walled carbon nanotubes by dynamic and static compression

We report on a first study of single walled carbon nanotubes (SWCNTs) after application of dynamic (shock) compression. The experiments were conducted at 19 GPa and 36 GPa in a recovery assembly. For comparison, an experiment at a static pressure of 36 GPa was performed on the material from the same batch in a diamond anvil cell (DAC). After the high pressure treatment the samples were characterized by Raman spectroscopy and transmission electron microscopy (TEM). After exposure to 19 GPa of shock compression the CNT material exhibited substantial structural damage such as CNT wall disruption, opening of the tube along its axis (“unzipping”) and tube shortening (“cutting”). Dynamic compression to 36 GPa resulted in essentially complete CNT destruction whereas at least a fraction of the nanotubes was recovered after 36 GPa of static compression though severely damaged. The results of these shock wave experiments underline the prospect of using SWCNTs as reinforcing units in material WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation both preserves the intrinsic membrane-defined directional quality of membrane proteins, and provides the mechanism for resonance line narrowing. The hydration-optimized samples overcome some of the difficulties associated with multi-dimensional, high-resolution, solid-state NMR spectroscopy of membrane proteins. These samples have greater stability over the course of multi-dimensional NMR experiments, they have lower sample conductance for greater rf power efficiency, and enable greater rf coil filling factors to be obtained for improved experimental sensitivity. Sample preparation is illustrated for the membrane protein CHIF (channel inducing factor), a member of the FXYD family of ion transport regulators.     

A Study for the Cause of Ferulic Acid-Induced Quenching of Tyrosine Fluorescence and Whether it is a Reliable Marker of Intermolecular Interactions or Not

Intrinsic fluorescence of peptides and proteins is extensively used to monitor their specific interactions with several natural and synthetic molecules known to have wide-ranging beneficial or detrimental effects on health. A consequence of these interactions would be a significant decrease of the fluorescence emission intensity of Tyrosine (Tyr) and/or Tryptophan (Trp) residues in the protein due to structural rearrangements of proteic microenvironment. However fluorescence quenching can be also caused by “trivial” artefacts. In this study we examined the effect of Ferulic acid (FA) on Tyr fluorescence. FA is a natural anti-oxidant suggested to bind to and to modify the structural properties of several proteins thus altering their biological activities. Fluorescence spectroscopy experiments on Tyr and on proteins containing Tyr and no Trp like beta amyloid peptides and Insulin were performed. Our results suggest that Tyr fluorescence loss can mainly result from an inner filter effect rather than from specific interactions with FA.     

Big entropy fluctuations in statistical equilibrium: The macroscopic kinetics

Large entropy fluctuations in the equilibrium steady state of classical mechanics are studied in extensive numerical experiments in a simple strongly chaotic Hamiltonian model with two degrees of freedom described by the modified Arnold cat map. The rise and fall of a large separated fluctuation is shown to be described by the (regular and stable) “macroscopic” kinetics, both fast (ballistic) and slow (diffusive). We abandon a vague problem of the “appropriate” initial conditions by observing (in a long run) a spontaneous birth and death of arbitrarily big fluctuations for any initial state of our dynamical model. Statistics of the infinite chain of fluctuations similar to the Poincaré recurrences is shown to be Poissonian. A simple empirical relationship for the mean period between the fluctuations (the Poincaré “cycle”) is found and confirmed in numerical experiments. We propose a new representation of the entropy via the variance of only a few trajectories (“particles”) that greatly facilitates the computation and at the same time is sufficiently accurate for big fluctuations. The relation of our results to long-standing debates over the statistical “irreversibility” and the “time arrow” is briefly discussed.     

19F固体核磁共振技术研究膜蛋白相互作用的进展

固体核磁共振技术因其可实现细胞膜环境中的蛋白质结构研究而广受关注.¹⁹F元素由于灵敏度高、天然丰度高,无生物背景等优点,被广泛应用于生物核磁共振技术中.氟标固体核磁共振技术常被用于细胞膜中蛋白质的相互作用研究,如：抗菌肽与细胞膜的相互作用、聚合膜蛋白结构分析等.此篇综述介绍了常用的蛋白质氟标修饰的实验方法,总结了常用的¹⁹F生物固体核磁共振实验技术,以及介绍了应用¹⁹F固体核磁共振研究膜蛋白的成功案例.此外,此篇综述讨论了¹⁹F固体核磁共振技术在蛋白质研究中的局限性.     

Acoustic disturbance from gas non-uniformities convected through a nozzle

The non-steady flow generated by convection of gas containing non-uniform temperature regions or “entropy spots” through a nozzle is examined analytically as a source of acoustic disturbance. The first portion of the investigation treats the “compact nozzle”, the case where all wave lengths are much longer than the nozzle. Strengths of transmitted and reflected one-dimensional waves are given for supersonic and subsonic nozzles and for one configuration of supersonic nozzle with normal shock at the outlet. In addition to a wave reflected from the nozzle inlet, the supersonic nozzle discharges two waves, one facing upstream and the other facing downstream. For reasonable values of the nozzle inlet Mach number, the pressure amplitude of each wave increases directly as the discharge Mach number.The acoustic perturbations from a supercritical nozzle of finite length, in which the undisturbed gas velocity increases linearly through the nozzle, are analyzed for several inlet and discharge Mach number values and over a wide frequency range. The results which agree with the compact analysis for low frequency, deviate considerably as the frequency rises, achieving pressure fluctuation levels of several times the compact values. It is shown that this result originates in a phase shift between the two waves emitted downstream and that the pressure fluctuations for moderate frequencies may be approximated from the compact analysis with an appropriate phase shift.In all cases, the pressure fluctuations caused by a 2% fluctuation in absolute inlet temperature are large enough to require consideration in acoustic analysis of nozzles or turbine blade channels.     

Effect of pressure on the electric transport properties of carbon-doped EuB6

We report about the influence of external pressure on the electrical resistivity of EuB_5.99C_0.01, the compound believed to be intrinsically inhomogeneous due to fluctuations of carbon content. Our results show that the low-temperature resistivity maximum shifts to a lower temperature with applied pressure, opposite to the behavior reported for stoichiometric EuB₆. The origin of such a qualitative difference we associate with the increasing volume fraction of the phase that is not compatible with ferromagnetic ordering (originating in regions with relatively higher carbon concentration) with increasing pressure. Our results support a recent proposition of Batkova et al. (2008) [12] that carbon-rich regions strongly influence magnetotransport properties of carbon-doped EuB₆, such as they play a role of “spacers”, which prevent the percolation of a ferromagnetic phase.     

核磁共振波谱在肿瘤诊疗中的应用研究进展

在介绍肿瘤样品代谢物的核磁共振波谱技术的研究方法的基础上,从离体组织和活体组织两个方面综述核磁共振波谱(NMR)在诊断肿瘤方面的应用进展,分析了它在肿瘤诊疗中的应用前景。在离体组织方面,人们通过1H和31P-NMR谱观测病人的体液样品、培养的细胞、切除的组织等来研究脂质、磷脂等代谢物的分布,观测肿瘤与对照组织之间的差别。其中利用组织提取物的方法能够得到分辨率较高的图谱,非常适合应用于肿瘤诊断和治疗方法的研究。高分辨魔角旋转(HR-MAS)的方法在肿瘤诊断研究方面展现出新的生命力,利用高分辨魔角旋转技术可以直接得到组织细胞中很多分子水平的代谢物结构和组成信息,因此它在癌症的早期诊断中具有很好的前景。在活体核磁共振波谱诊断肿瘤方面,主要应用1H和31P核磁共振波谱,结合MRI为非侵入性肿瘤分析提供了一种临床可用的方法。MRI与MRS技术的结合将使核磁共振波谱在医学领域有更大的应用空间。