磁共振波谱时域量化方法研究

磁共振波谱(MRS)信号可以在时域中采用全自动的暗箱方法或交互式方法进行量化。介绍了这两种方法的运算法则,并对MRS波谱数据进行了处理,得到了很好的结果。     

Dispersive Raman spectroscopy allows the identification and quantification of melanin types

Melanins are the most prevalent pigments in animals and are involved in visual communication by producing colored traits that often evolve as intraspecific signals of quality. Identifying and quantifying melanins are therefore essential to understand the function and evolution of melanin‐based signals. However, the analysis of melanins is difficult due to their insolubility and the lack of simple methods that allow the identification of their chemical forms. We recently proposed the use of Raman spectroscopy as a simple, noninvasive technique that can be used to identify and quantify melanins in feathers and hairs. Contrarily, other authors later stated that melanins are characterized by a lack of defined Raman signals. Here, we use confocal Raman microscopy to confirm previous analyses showing that the two main chemical forms of melanins (eumelanin and pheomelanin) exhibit distinct Raman signal and compare different excitation wavelengths to analyze synthetic pheomelanin and natural melanins in feathers of different species of birds. Our analyses indicate that only laser excitation wavelengths below 1064 nm are useful for the analysis of melanins by Raman spectroscopy, and only 780‐nm laser in the case of melanins in feathers. These findings show that the capacity of Raman spectroscopy to distinguish different chemical forms of melanins depends on laser power and integration time. As a consequence, Raman spectroscopy should be applied after preliminar analyses using a range of these parameters, especially in fragile biological tissues such as feathers.     

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.     

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in 1H nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.     

Modelling noninvasively measured cerebral signals during a hypoxemia challenge: steps towards individualised modelling

Noninvasive approaches to measuring cerebral circulation and metabolism are crucial to furthering our understanding of brain function. These approaches also have considerable potential for clinical use "at the bedside". However, a highly nontrivial task and precondition if such methods are to be used routinely is the robust physiological interpretation of the data. In this paper, we explore the ability of a previously developed model of brain circulation and metabolism to explain and predict quantitatively the responses of physiological signals. The five signals all noninvasively-measured during hypoxemia in healthy volunteers include four signals measured using near-infrared spectroscopy along with middle cerebral artery blood flow measured using transcranial Doppler flowmetry. We show that optimising the model using partial data from an individual can increase its predictive power thus aiding the interpretation of NIRS signals in individuals. At the same time such optimisation can also help refine model parametrisation and provide confidence intervals on model parameters. Discrepancies between model and data which persist despite model optimisation are used to flag up important questions concerning the underlying physiology, and the reliability and physiological meaning of the signals.     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy

Biological and pharmacological interactions of heparin and structurally related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues. Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by high-performance liquid chromatographic analysis of fragments obtained by enzymatic or chemical degradation. Nuclear magnetic resonance (NMR) spectroscopy is also currently used for structural characterization of GAGs. However, the use of monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not allow reliable quantitative measurements. Using magnetically equivalent signals, the higher resolution achieved by two-dimensional NMR methods could be also exploited for quantitative applications. In this work, heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and HS mimics obtained by chemical modification of the Escherichia coli K5 polysaccharide (K5-PS) structurally related to the common biosynthetic precursor of heparin and HS. Heparin was used as a model for assessing the influence of 1H-13C spin-spin couplings on "volumes" of the corresponding signals. For major signals, the HSQC approach permitted quantification of additional structural features both in heparins and in a typical HS. The method was applied to profile the substitution patterns of K5-PS derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.     

Structural features of the protoporphyrin-apomyoglobin complex: a proton NMR spectroscopy study

The structural properties of the complex formed by apomyoglobin and protoporphyrin IX (des-iron myoglobin) were studied to probe the influence of iron-to-histidine coordination on the native myoglobin fold and the heme binding site geometry. Standard two-dimensional proton nuclear magnetic resonance spectroscopy methods were applied to identify porphyrin and protein signals. A pronounced spectral resemblance between carbonmonoxymyoglobin and des-iron myoglobin was noticed that could be exploited to assign a number of resonances by nuclear Overhauser spectroscopy. Protoporphyrin IX was determined to bind in the same orientation as the heme. Most residues in contact with the prosthetic group were found in the holomyoglobin conformation. Several tertiary structure features were also characterized near the protein termini. It was concluded that the protoporphyrin-apomyoglobin interactions are capable of organizing the binding site and the unfolded region of the apoprotein into the native holoprotein structure.     

Bimodal quantitative monitoring for enzymatic activity with simultaneous signal increases in 19F NMR and fluorescence using silica nanoparticle-based molecular probes

We describe the bimodal quantitative assay for enzymatic activity in (19)F NMR spectroscopy and fluorescence spectroscopy using a nanoparticle-based molecular probe. Perfluorinated dendrimers were tethered on silica nanoparticles with a phosphate-caged fluorescein as a linker. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers should be highly restricted, and the (19)F NMR signals from the perfluorinated dendrimers were too broad to be detected relative to the noise level. Fluorescence signals of fluorescein were suppressed by the presence of the diphosphate groups. Following the enzymatic reaction with an alkaline phosphatase, perfluorinated dendrimers and fluorescein were released, and the NMR signals of perfluorinated dendrimers and strong fluorescence from fluorescein were correspondingly observed. The enzymatic activity and reaction rates of the hydrolysis of alkaline phosphatase were detected from the increases of fluorescence and (19)F NMR signals. Finally, the feasibility of the probe in the presence of miscellaneous molecules under biomimetic conditions was demonstrated by determining of the enzymatic activity in cell lysate. Quantitative analysis using both (19)F NMR spectroscopy and fluorescence spectroscopy can be accomplished.     

Solution structural characteristics of cyanometmyoglobin: resonance assignment of heme cavity residues by two-dimensional NMR

Steady-state nuclear Overhauser effects (NOE), two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY), and 2D spin correlation spectroscopy (COSY) have been applied to the fully paramagnetic low-spin, cyanide-ligated complex of sperm whale ferric myoglobin to assign the majority of the heme pocket side-chain proton signals and the remainder of the heme signals. It is shown that the 2D NOESY map reveals essentially all dipolar connectivities observed in ordinary 1D NOE experiments and expected on the basis of crystal coordinates, albeit often more weakly than in a diamagnetic analogue. For extremely broad (approximately 600-Hz) and rapidly relaxing (Tf1 approximately 3 ms) signals which show no NEOSY peaks, we demonstrate that conventional steady-state NOEs obtained under very rapid pulsing conditions still allow detection of the critical dipoar connectivities that allow unambiguous assignments. The COSY map was found to be generally less useful for the hyperfine-shifted residues, with cross peaks detected only for protons greater than 6 A from the iron. Nevertheless, numerous critical COSY cross peaks between strongly hyperfine-shifted peaks were resolved and assigned. In all, 95% (53 of 56 signals) of the total proton sets within approximately 7.5 A of the iron, the region experiencing the strongest hyperfine shifts and paramagnetic relaxation, are now unambiguously assigned. Hence it is clear that the 2D methods can be profitably applied to paramagnetic proteins. The scope and limitations of such application are discussed. The resulting hyperfine shift pattern for the heme confirmed expectations based on model compounds. In contrast, while exhibiting fortuitous 1H NMR spectral similarities, a major discrepancy was uncovered between the hyperfine shift pattern of the axially bound (F8 histidyl) imidazole in the protein and that of the imidazole in a relevant model compound [Chacko, V.P., & La Mar, G. N. (1982) J. Am. Chem. Soc. 104, 7002-7007], providing direct evidence for a protein-based deformation of axial bonding in the protein.     

Proton transfer in the photoreceptors phytochrome and photoactive yellow protein.

Light-induced activation of the photoreceptors phytochrome and photoactive yellow protein (PYP) is accompanied by protonation changes of the respective chromophores and key residues in the protein moiety. For both systems, proton exchange with the external medium could be observed with pH electrode measurements and with UV-visible absorption spectroscopy using appropriate pH indicator dyes. From these signals, the stoichiometry of proton release and uptake, respectively, was determined by different calibration procedures which will be presented and discussed. Kinetic information on these processes is only available from time-resolved measurements with pH indicator dyes. Vibrational spectroscopy methods such as Fourier transform infrared spectroscopy and resonance Raman scattering provided information on the protonation state of individual functional groups suggesting that internal proton transfer processes are involved as well. Deuterium kinetic isotope effects that occurred in the Pr --> Pfr phototransformation of the bacteriophytochromes Cph1 and Agp1 were consistent with proton transfer reactions as rate-limiting steps. In contrast, the apparent rate constants in the photocycle of PYP exhibited only small kinetic isotope effects that could not be interpreted conclusively. Possible mechanisms of proton transfer in the activation of phytochrome and PYP will be discussed.     

The use of nanoparticles for the study of conformations submembrane hemoglobin

The advantages and features of the integrated application of methods of atomic force microscopy, laser interference microscopy and Raman microscopy in the study of erythrocytes was discussed. For the successful application of Raman microscopy in surface enhanced Raman spectroscopy mode the silver colloids was using. The dependence of the enhancement of Raman signals from silver colloids size was demonstrated. The using of developed methods for clinic diagnostic was discussed.     

Classification of lactic acid bacteria with UV-resonance Raman spectroscopy

UV-resonance Raman spectroscopy is applied as a method for the identification of lactic acid bacteria from yogurt. Eight different strains of bacteria from Lactobacillus acidophilus, L. delbrueckii ssp. bulgaricus, and Streptococcus thermophilus were investigated. At an excitation wavelength of 244 nm signals from nucleic acids and proteins are selectively enhanced. Classification was accomplished using different chemometric methods. In a first attempt, the unsupervised methods hierarchical cluster analysis and principal component analysis were applied to investigate natural grouping in the data. In a second step the spectra were analyzed using several supervised methods: K-nearest neighbor classifier, nearest mean classifier, linear discriminant analysis, and support vector machines.     

Direct visualization and profiling of protein misfolding and aggregation in live cells

Over the past few years, research tools have been developed to monitor the multistep protein aggregation process in live cells, a process that has been associated with a growing number of human diseases. Herein, we describe recent advances in methods that can either survey the distribution of aggregation at the level of the cellular proteome using mass spectroscopy or discern the multistep aggregation process of specific proteins of interest via fluorescence signals. Future development and application of such technologies are expected to provide insights on mechanisms, diagnosis, and treatment of diseases rooted in protein aggregation.     

NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase from Phanerochaete chrysosporium. Comparison with horseradish peroxidase

One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting state and the low spin, cyanide-inhibited derivative of isozyme H2 of the lignin peroxidase, LiP, from Phanerochaete chrysosporium strain BKM 1767. One-dimensional NMR revealed a resting state LiP that is five coordinate at 25 degrees C with an electronic structure similar to that of horseradish peroxidase, HRP. Differential paramagnetic relaxivity was used to identify the C beta H signals of the axial His177. A combination of bond correlation spectroscopy and nuclear Overhauser effect spectroscopy of cyanide-inhibited LiP (LiP-CN) has allowed the assignment of all resolved heme resonances without recourse to isotope labeling, as well as those of the proximal His177 and the distal His48. The surprising effectiveness of the two dimensional NMR methods on such a large and paramagnetic protein indicates that such two dimensional experiments can be expected to have major impact on solution structure determination of diverse classes of heme peroxidases. The two dimensional NMR data of LiP-CN reveal a heme contact shift pattern that reflects a close similarity to that of HRP-CN, including the unusual in-plane trans and cis orientation of the 2- and 4-vinyls. The axial His177 also exhibits the same orientation relative to the heme as in HRP-CN. The proximal His177 contact shifted resonances of both the low spin LiP-CN and high spin LiP are shown to reflect significantly reduced hydrogen bond donation by, or imidazolate character for, the axial histidine in LiP relative to HRP, which may explain the higher redox potential of LiP. The signals are identified for a distal residue that originates from the protonated His48 with disposition relative to the heme similar to that found for the distal His42 in HRP-CN. In contrast, the absence of any resolved signals attributable to an Arg44 in LiP-CN suggest that this distal residue has an altered orientation relative to the heme compared with that of the conserved Arg38 in HRP-CN (Thanabal, V., de Ropp, J. S., and La Mar, G. N. (1987) J. Am. Chem. Soc. 109, 7516-7525).     

Relaxation filtered hyperfine (REFINE) spectroscopy: a novel tool for studying overlapping biological electron paramagnetic resonance signals applied to mitochondrial complex I

A simple strategy to separate overlapping electron paramagnetic resonance (EPR) signals in biological systems is presented. Pulsed EPR methods (inversion- and saturation-recovery) allow the determination of the T(1) spin-lattice relaxation times of paramagnetic centers. T(1) may vary by several orders of magnitude depending on the species under investigation. These variations can be employed to study selectively individual species from a spectrum that results from an overlap of two species using an inversion-recovery filtered (IRf) pulsed EPR technique. The feasibility of such an IRf field-swept technique is demonstrated on model compounds (alpha,gamma-bisphenylene-beta-phenylallyl-benzolate, BDPA, and 2,2,6,6-tetramethyl-piperidine-1-oxyl, TEMPO) and a simple strategy for the successful analysis of such mixtures is presented. Complex I is a multisubunit membrane protein of the respiratory chain containing several iron-sulfur (FeS) centers, which are observable with EPR spectroscopy. It is not possible to investigate the functionally important FeS cluster N2 separately because this EPR signal always overlaps with the other FeS signals. This cluster can be studied selectively using the IRf field-swept technique and its EPR spectrum is in excellent agreement with previous cw-EPR data from the literature. In addition, the possibility to separate the hyperfine spectra of two spectrally overlapping paramagnetic species is demonstrated by applying this relaxation filter together with hyperfine spectroscopy (REFINE). For the first time, the application of this filter to a three-pulse electron spin-echo envelope modulation (ESEEM) pulse sequence is demonstrated to selectively observe hyperfine spectra on a system containing two paramagnetic species. Finally, REFINE is used to assign the observed nitrogen modulation in complex I to an individual iron-sulfur cluster.     

Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction.

The spectroscopic methods of fluorescence polarization and electron paramagnetic resonance (EPR) are used to study order and orientation of extrinsically labeled protein elements of ordered biological systems. These methods generate complementary information about the order of the system, but a consistent quantitative interpretation of the related data is complicated because the signals arise from different donors. We introduce a new method that allows us to detect both signals from the same donor. Unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction, the quinoidal structure of the xanthene ring is transformed into a semiquinone, and a pi-radical is formed having a characteristic EPR signal of an unpaired electron spin with proton hyperfine interactions. A strong EPR signal is observed from the dye in solution or when specifically attached to a protein following irradiation in the presence of dithiothreitol or cysteine. We applied this technique to the study of skeletal muscle fibers. The fluorescent dye (iodoacetamido)fluorescein was covalently attached to the reactive thiol of the myosin molecule in muscle fibers. Fluorescence polarization and EPR spectroscopy were performed on the labeled fibers in rigor. Both signals indicate a highly ordered system characteristic of cross-bridges bound to actin. Our use of the same signal donor for fluorescence and EPR studies of probe order is a promising new technique for the study of order in protein elements of biological assemblies.     

Flash-induced kinetic infrared spectroscopy applied to biochemical systems

A flash photolysis apparatus with monitoring infrared beam is described allowing measurements of relative transmission changes of 10^–3 in times of a few milliseconds. The investigation of the photodissociation of CO-myoglobin confirms the results obtained by static infrared difference spectroscopy. The application of our method to the rhodopsin/Meta II transition reveals signals which can tentatively be ascribed to the disappearance of the C=C-band of the protonated N-retinylidene Schiff base in rhodopsin. The developed method will be compared with other existing methods of kinetic vibronic spectroscopy such as kinetic resonance Raman spectroscopy and kinetic Fourier infrared spectroscopy.