同步辐射圆二色谱及其在糖生物学及生物分子中的应用

同步辐射圆二色谱与普通圆二色谱相比,特点在于向真空紫外波段（〈200nm）拓展,以及同步辐射所提供的高强度紫外和真空紫外光源。糖的圆二色谱结构主要在200nm以下。蛋白质和核酸在200nm以下的真空紫外范围,也具有丰富的光谱结构。因此向真空紫外拓展,伴随新的电子跃迁,对应新的光谱结构,包含更丰富的结构信息,确定的结构种类就越多和越精确。同步辐射高强度的真空紫外光源,是获得高质量真空紫外圆二色谱数据的保证,为糖及糖蛋白、蛋白质和核酸研究提供了溶液中结构探测新的实验方法。综述同步辐射圆二色谱特点及其在结构生物学中的应用,以及新发展的蛋白质圆二色谱数据库（PCDDB）。介绍已对外开放的北京同步辐射实验室同步辐射圆二色谱探测,及其在蛋白质、糖和核酸研究中的应用,以及基于微流控混合芯片的亚毫秒动态探测发展。     

Comprehensive secondary‐structure analysis of disulfide variants of lysozyme by synchrotron‐radiation vacuum‐ultraviolet circular dichroism

To elucidate the effects of specific disulfide bridges (Cys6‐Cys127, Cys30‐Cys115, Cys64‐Cys80, and Cys76‐Cys94) on the secondary structure of hen lysozyme, the vacuum‐ultraviolet circular dichroism (VUVCD) spectra of 13 species of disulfide‐deficient variants in which Cys residues were replaced with Ala or Ser residues were measured down to 170 nm at pH 2.9 and 25°C using a synchrotron‐radiation VUVCD spectrophotometer. Each variant exhibited a VUVCD spectrum characteristic of a considerable amount of residual secondary structures depending on the positions and numbers of deleted disulfide bridges. The contents of α‐helices, β‐strands, turns, and unordered structures were estimated with the SELCON3 program using the VUVCD spectra and PDB data of 31 reference proteins. The numbers of α‐helix and β‐strand segments were also estimated from the VUVCD data. In general, the secondary structures were more effectively stabilized through entropic forces as the number of disulfide bridges increased and as they were formed over larger distances in the primary structure. The structures of three‐disulfide variants were similar to that of the wild type, but other variants exhibited diminished α‐helices with a border between the ordered and disordered structures around the two‐disulfide variants. The sequences of the secondary structures were predicted for all the variants by combining VUVCD data with a neural‐network method. These results revealed the characteristic role of each disulfide bridge in the formation of secondary structures. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Synchrotron radiation circular dichroism spectroscopy applied to metmyoglobin and a 4-alpha-helix bundle carboprotein

The novel technique, synchrotron radiation-based circular dichroism (SR-CD), has been applied to the study of metmyoglobin and a carboprotein (carbohydrate-based peptide with protein tertiary structure) with 4-alpha-helix bundle structure, as well as a carbopeptide (carbohydrate-based peptide) with a truncated peptide sequence. The use of synchroton radiation (SR) enabled circular dichroism (CD) measurements in the vacuum ultraviolet (VUV) down to 168 nm in D(2)O and 160 nm in 2,2,2-trifluoroethanol (TFE). The band shape in the CD spectra in the low wavelength region was studied, comparing samples with two types of alpha-helical tertiary structure, namely the globin fold and the 4-alpha-helix bundle motif. No significant differences were found between the CD spectra of the alpha-helical samples (metmyoglobin and carboprotein) in D(2)O solution. The use of 2,2,2-TFE (TFE) as solvent clearly alters the VUV CD but the two samples have very similar CD spectra. The solvent-induced denaturing of metmyoglobin in TFE was observed using absorption and CD spectroscopy of the Soret band, with results indicating heme release. The VUV spectrum of TFE-denatured metmyoglobin exhibits dramatic differences in comparison with previous studies of the native enzyme in aqueous solution. The implications of this observation are discussed.     

Analyses of circular dichroism spectra of membrane proteins

Circular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.     

Formation of G‐wires,bimolecular and tetramolecular quadruplex: Cation‐induced structural polymorphs of G‐rich DNA sequence of human SYTX gene

An exceptional property of auto‐folding into a range of intra‐ as well as intermolecular quadruplexes by guanine‐rich oligomers (GROs) of promoters, telomeres and various other genomic locations is still one of the most attractive areas of research at present times. The main reason for this attention is due to their established in vivo existence and biological relevance. Herein, the structural status of a 20‐nt long G‐rich sequence with two G5 stretches (SG20) is investigated using various biophysical and biochemical techniques. Bioinformatics analysis suggested the presence of a 17‐nt stretch of this SG20 sequence in the intronic region of human SYTX (Synaptotagmin 10) gene. The SYTX gene helps in sensing out the Ca²⁺ ion, causing its intake in the pre‐synaptic neuron. A range of various topologies like bimolecular, tetramolecular and guanine‐wires (nano‐wires) was exhibited by the studied sequence, as a function of cations (Na⁺/K⁺) concentration. UV‐thermal denaturation, gel electrophoresis, and circular dichroism (CD) spectroscopy showed correlations and established a cation‐dependent structural switch. The G‐wire formation, in the presence of K⁺, may further be explored for its possible relevance in nano‐biotechnological applications.     

Secondary-structure analysis of alcohol-denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structural characteristics of alcohol-denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra of six proteins-myoglobin, human serum albumin, α-lactalbumin, thioredoxin, β-lactoglobulin, and α-chymotrypsinogen A-down to 170 nm in trifluoroethanol solutions (TFE: 0-50%) and down to 175 nm in methanol solutions (MeOH: 0-70%) at pH 2.0 and 25°C, using a synchrotron-radiation VUVCD spectrophotometer. The contents of α-helices, β-strands, turns, poly-L-proline type II helices (PPIIs), and unordered structures of these proteins were estimated using the SELCON3 program, including the numbers of α-helix and β-strand segments. Furthermore, the positions of α-helices and β-strands on amino acid sequences were predicted by combining these secondary-structure data with a neural-network method. All alcohol-denatured proteins showed higher α-helix contents (up to ~ 90%) compared with the native states, and they consisted of several long helical segments. The helix-forming ability was higher in TFE than in MeOH, whereas small amounts of β-strands without sheets were formed in the MeOH solution. The produced α-helices were transformed dominantly from the β-strands and unordered structures, and slightly from the turns. The content and mean length of α-helix segments decreased as the number of disulfide bonds in the proteins increased, suggesting that disulfide bonds suppress helix formation by alcohols. These results demonstrate that alcohol-denatured proteins constitute an ensemble of many long α-helices, a few β-strands and PPIIs, turns, and unordered structures, depending on the types of proteins and alcohols involved.     

Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data

The effects of spectral magnitude on the calculated secondary structures derived from circular dichroism (CD) spectra were examined for a number of the most commonly used algorithms and reference databases. Proteins with different secondary structures, ranging from mostly helical to mostly beta-sheet, but which were not components of existing reference databases, were used as test systems. These proteins had known crystal structures, so it was possible to ascertain the effects of magnitude on both the accuracy of determining the secondary structure and the goodness-of-fit of the calculated structures to the experimental data. It was found that most algorithms are highly sensitive to spectral magnitude, and that the goodness-of-fit parameter may be a useful tool in assessing the correct scaling of the data. This means that parameters that affect magnitude, including calibration of the instrument, the spectral cell pathlength, and the protein concentration, must be accurately determined to obtain correct secondary structural analyses of proteins from CD data using empirical methods.     

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.     

Distinct circular dichroism spectroscopic signatures of polyproline II and unordered secondary structures: Applications in secondary structure analyses

Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically‐defined spectroscopic signatures derived from proteins with known three‐dimensional structures. Many proteins identified as being “Intrinsically Disordered Proteins” have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as “other”, “random coil”, “unordered”, or “disordered”. However the “other” category can also include polyproline II (PPII)‐type structures, whose spectral properties have not been well‐distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII‐type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid‐containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins.     

Vacuum-ultraviolet circular dichroism study of saccharides by synchrotron radiation spectrophotometry

Vacuum-ultraviolet circular dichroism (VUVCD) spectra of five monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, and D-lyxose) and five disaccharides (maltose, isomaltose, cellobiose, gentiobiose, and lactose) were measured to 160 nm using a synchrotron-radiation VUVCD spectrophotometer in aqueous solution under high vacuum at 25 degrees C. Most of the saccharides show a positive peak with some shoulders at around 170 nm, except for D-galactose and lactose, which show two distinct negative peaks at around 165 and 177 nm. These spectra are influenced by such structural factors as alpha and beta anomers at C-1, axial and equatorial hydroxyl groups at C-2 and C-4, trans (T) and gauche (G) conformations of the hydroxymethyl group at C-5, and the type of glycosidic linkage. Deconvolution of the VUVCD spectra of D-glucose, D-mannose, and D-galactose into six independent Gaussian components for alpha-GG, alpha-GT, alpha-TG, beta-GG, beta-GT, and beta-TG conformations suggests that the alpha anomer has red-shifted spectra relative to the beta anomer, and that GG and GT conformations have positive and negative circular dichroism signs, respectively, while the sign for TG conformation is anomer dependent. These speculations from the deconvolution analyses are also supported by the VUVCD spectra of disaccharides. These results give new insight into the equilibrium conformations of saccharides, demonstrating the usefulness of synchrotron-radiation VUVCD spectroscopy.     

Conformation of membrane‐bound proteins revealed by vacuum‐ultraviolet circular‐dichroism and linear‐dichroism spectroscopy

Knowledge of the conformations of a water‐soluble protein bound to a membrane is important for understanding the membrane‐interaction mechanisms and the membrane‐mediated functions of the protein. In this study we applied vacuum‐ultraviolet circular‐dichroism (VUVCD) and linear‐dichroism (LD) spectroscopy to analyze the conformations of α‐lactalbumin (LA), thioredoxin (Trx), and β‐lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural‐network analysis showed that these three proteins have characteristic helix‐rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence‐level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane‐binding proteins, which are difficult targets for X‐ray crystallography and nuclear magnetic resonance spectroscopy. Proteins 2016; 84:349–359. © 2016 Wiley Periodicals, Inc.     

ILPR repeats adopt diverse G‐quadruplex conformations that determine insulin binding

The insulin‐linked polymorphic region (ILPR) is a VNTR region located upstream of the insulin (INS) gene consisting of the repeat 5′‐ACAGGGGTGTGGGG (repeat a) and several less abundant sequence repeats (b–n). Here, we have investigated the structural polymorphism of G‐quadruplexes formed from the most common repeat sequences (a–c) and their effect on insulin protein binding. We first established that the ILPR repeats “b” and “c” can form quadruplex structures. Insulin has previously been shown to bind a G‐quadruplex formed by a dimer of the repeat “a”. Our findings show that insulin binds preferentially to the repeat “a” G‐quadruplex (K_d = 0.17 ± 0.03 μM) over G‐quadruplexes formed from other ILPR repeats that were tested (K_ds from 0.71 ± 0.15 to 1.07 ± 0.09 μM). Additionally, the Watson‐Crick complementary relationship between the loop regions of repeat “a” (ACA and TGT) seemingly play an important role in favoring a specific G‐quadruplex conformation, which based on our data is critical for insulin binding. Affinity for insulin is reduced in sequences lacking the putative WC complementarity, however upon engineered restoration of complementarity, insulin binding is recovered. A DMS footprinting assay on the repeat “a” G‐quadruplex in the presence of insulin, combined with binding affinities for ILPR mutants led to identification of a loop nucleotide critical for binding. Uniquely, insulin shows clear preference for binding to the G‐quadruplexes with the more antiparallel feature. Collectively, our results illustrate the specific nature of insulin binding to the ILPR G‐quadruplexes and begin to provide molecular details on such interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 21–31, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Folding thermodynamics of the hybrid‐1 type intramolecular human telomeric G‐quadruplex

Guanine‐rich DNA sequences that may form G‐quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G‐quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G‐quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G‐rich sequences in the presence or the absence of their complementary C‐rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26‐meric human telomeric sequence d[A₃G₃(T₂AG₃)₃A₂]. In the presence of K⁺ ions, the latter adopts the hybrid‐1 G‐quadruplex conformation, a tightly packed structure with an unusually small number of solvent‐exposed atomic groups. The K⁺‐induced folding of the G‐quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G‐quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G‐quadruplex formation. Based on our volume data, 432 ± 19 water molecules become released to the bulk upon the G‐quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute–solvent interactions all over the surface of the folded structure. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 216–227, 2014.     

Structural analysis of lysine‐4 methylated histone H3 proteins using synchrotron radiation circular dichroism spectroscopy

We report structural alterations of histone H3 proteins induced by lysine‐4 (K4) monomethylation, dimethylation, and trimethylation identified by using synchrotron radiation circular dichroism spectroscopy. Compared with unmethylated H3, monomethylation and dimethylation induced increases in α‐helix structures and decreases in β‐strand structures. In contrast, trimethylation decreased α‐helix content but increased β‐strand content. The structural differences among K4‐unmethylated/methylated H3 may allow epigenetic enzymes to discriminate the substrates both chemically and sterically.     

Electronic structures of azulene-fused porphyrins as seen by magnetic circular dichroism and TD-DFT calculations

A combination of magnetic circular dichroism (MCD), electronic absorption spectroscopy and time-dependent density functional theory (TD-DFT) calculations has been used to investigate the electronic structure of azulene-fused pi-expanded porphyrins based primarily on the spectral properties of absorption bands in the near infrared region. From MCD experiments, it was suggested that in the case of a mono-azulene-fused porphyrin DeltaHOMO approximately equal DeltaLUMO (where DeltaHOMO is the magnitude of the energy gap between the HOMO and HOMO-1 and DeltaLUMO is the magnitude of the energy gap between the LUMO and LUMO+1), while in the case of an oppositely-di-azulene-fused porphyrin, DeltaHOMO相似文献   

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Absolute configuration assignment of (+)‐fluralaner using vibrational circular dichroism

The absolute configurations of the separated enantiomers of fluralaner, a racemic animal health product used to prevent fleas and ticks, have been assigned using vibrational circular dichroism (VCD). The crystallographic structure of the active enantiomer (+)‐fluralaner has previously been shown to have the (S ) configuration using small molecule crystallography. We sought a faster analytical method to determine the absolute configuration of the separated enantiomers. When comparing the measured IR (infrared) and VCD spectra, it is apparent that the amide carbonyl groups appear in the IR but are nearly absent in the VCD. Computational work to calculate the VCD and IR using in vacuo models, implicit solvation, and explicitly solvated complexes has implicated conformational averaging of the carbonyl VCD intensities.     

Absolute configuration of (−)-myrtenal by vibrational circular dichroism

The VCD spectrum of the monoterpene (−)-myrtenal (1) was compared with theoretical spectra using ab initio density functional theory (DFT) calculations at the B3LYP/6-31G(d,p), B3LYP/6-31G+(d,p), B3LYP/6-311G+(d,p), B3LYP/DGDZVP, and B3PW91/DGTZVP levels of theory. Conformational analysis of 1 indicated that the lowest energy conformer was s-trans-C2-C10, which contributes more than 98.5% to the total conformational population regardless of the employed level of theory. The use of a recently developed confidence level algorithm demonstrated that VCD spectra calculated for the main conformer, using the indicated hybrid functionals and basis set, gave no significant changes, from where it follows that B3LYP/DGDZVP calculations provide a superior balance between computer cost and VCD spectral accuracy. The DGDZVP basis set demanded around a quarter the time than the 6-311G+(d,p) basis set while providing similar results. The spectral comparison also provided evidence that the levorotatory enantiomer of myrtenal has the 1R absolute configuration.     

FANCJ promotes DNA synthesis through G‐quadruplex structures

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ^−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.