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1.
《Analytical letters》2012,45(5):855-873
Abstract A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrPc). After five rounds of panning against recombinant bovine prion protein (rb‐PrPc) and ELISA test, two positive clones with high affinity to rb‐PrPc, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrPc were 3.24×10?8 M, 8.82×10?8M, and 8.10×10?9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrPc with detection limit of 1 pg/ml. 相似文献
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3.
With an objective to develop β‐amyloid destabilizing agents, we have investigated the interactions of a few water‐soluble near‐infrared (NIR)‐absorbing squaraine dyes 1 – 3 with lysozyme and its amyloid aggregates through photophysical and biophysical techniques. These dyes exhibited strong interactions with lysozyme and β‐amyloids in addition to serum albumins as evidenced by the absorption and emission changes. The interactions were found to be spontaneous with association constant values in the range of approximately 104–105 m ?1, as confirmed through half‐reciprocal analysis and isothermal calorimetric measurements. Uniquely, such effective interactions of the dyes have led to the complete disassembly of the β‐amyloid fibrillar structures to form spherical particles approximately 350 nm in size, as confirmed through photophysical, thioflavin assay, circular dichroism (CD), atomic force microscopy (AFM), TEM, and selected‐area electron diffraction (SAED) techniques. These results demonstrate that the squaraine dyes 1 – 3 under investigation act as effective protein‐labelling and destabilizing agents of the protein amyloidogenesis. 相似文献
4.
Interdiction in the Early Folding of the p53 DNA-Binding Domain Leads to Its Amyloid-Like Misfolding
In this article, we investigate two issues: (a) the initial contact formation events along the folding pathway of the DNA-binding domain of the tumor suppressor protein p53 (core p53); and (b) the intermolecular events leading to its conversion into a prion-like form upon incubation with peptide P8(250-257). In the case of (a), the calculations employ the sequential collapse model (SCM) to identify the segments involved in the initial contact formation events that nucleate the folding pathway. The model predicts that there are several possible initial non-local contacts of comparative stability. The most stable of these possible initial contacts involve the protein segments 159AMAIY163 and 251ILTII255, and it is the only native-like contact. Thus, it is predicted to constitute “Nature’s shortcut” to the native structure of the core domain of p53. In the case of issue (b), these findings are then combined with experimental evidence showing that the incubation of the core domain of p53 with peptide P8(250-257), which is equivalent to the native protein segment 250PILTIITL257, leads to an amyloid conformational transition. It is explained how the SCM predicts that P8(250-257) effectively interdicts in the formation of the most stable possible initial contact and, thereby, disrupts the subsequent normal folding. Interdiction by polymeric P8(250-257) seeds is also studied. It is then hypothesized that enhanced folding through one or several of the less stable contacts could play a role in P8(250-257)-promoted core p53 amyloid misfolding. These findings are compared to previous results obtained for the prion protein. Experiments are proposed to test the hypothesis presented regarding core p53 amyloid misfolding. 相似文献
5.
Interaction of PiB‐Derivative Metal Complexes with Beta‐Amyloid Peptides: Selective Recognition of the Aggregated Forms
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Dr. André F. Martins David M. Dias Dr. Jean‐François Morfin Dr. Sara Lacerda Dr. Douglas V. Laurents Dr. Éva Tóth Prof. Carlos F. G. C. Geraldes 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(14):5413-5422
Metal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aβ1–40 peptide. We have studied lanthanide(III) chelates of two PiB‐derivative ligands (PiB=Pittsburgh compound B), L1 and L2, differing in the length of the spacer between the metal‐complexing DO3A macrocycle (DO3A= 1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic acid) and the peptide‐recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aβ1–40 (KD=67–160 μM ), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the 15N‐labeled, monomer Aβ1–40 peptide indicates nonsignificant interaction with monomeric Aβ. Time‐dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aβ1–40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aβ fibril formation distinctly. Whereas GdL1, at higher concentrations, stabilizes β‐sheets, GdL2 prevents aggregation by promoting α‐helical structures. These results give insight into the behavior of amyloid‐targeted metal complexes in general and contribute to a more rational design of metal‐based diagnostic and therapeutic agents for amyloid‐ associated pathologies. 相似文献
6.
AD (Alzheimer’s disease) is a progressive neurodegenerative disorder characterized by the cerebral accumulation of fibrillar amyloid‐beta (Aβ) aggregates. Here we present the electrochemistry of two novel sym‐triazine derivatives (TAE‐1, TAE‐2) as modulators of Aβ1–42 aggregation in vitro. Incubation studies conducted at physiological conditions demonstrated strong inhibition of β‐sheet fibril formation. Uniquely, square‐wave voltammetry indicated progressive changes in the surface‐availability of amyloid‐intercalated triazines for oxidation, mediated by competing peptide self‐assembly. Time‐resolved voltammetric analysis showed increasing anodic peak currents (≥3‐fold) and progressive shifts in redox potentials, measured over 24 h. The more potent aggregation modulator (TAE‐2) showed prolonged association during the pre‐nucleation states of Aβ. 相似文献
7.
Cross‐β Amyloid Nanohybrids Loaded With Cytochrome C Exhibit Superactivity in Organic Solvents
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Nidhi Kapil Ashmeet Singh Dr. Dibyendu Das 《Angewandte Chemie (International ed. in English)》2015,54(22):6492-6495
The present study reports the development of a unique class of Cytochrome C (CytC)‐loaded cross‐beta amyloid nanohybrids. The peroxidase activity of the bound CytC increased up to two orders of magnitude in organic solvents compared to the activity of unbound CytC in water. The amyloid sequences used in the study feature the nucleating core 17LVFF21 of the beta amyloid (Aβ), which assembled to form homogenous fibers and nanotubes. The morphology and exposed surface of the amyloid nanohydrids critically modulated the CytC activity. A CytC–Ac‐KLVFFAE‐NH2 hybrid featuring nanofiber morphology showed 308‐fold higher activity than unbound CytC in water, which increased to 450‐fold with the nanotube morphology of CytC–Ac‐KLVFFA L ‐NH2. Notably, activity declined substantially when the exposed surface charge was detuned by replacing lysine with histidine, thus underpinning the importance of surface charge. This enzyme–amyloid nanohybrid system could facilitate the technological application of enzymes. 相似文献
8.
Rational Design and Identification of a Non‐Peptidic Aggregation Inhibitor of Amyloid‐β Based on a Pharmacophore Motif Obtained from cyclo[‐Lys‐Leu‐Val‐Phe‐Phe‐]
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Tadamasa Arai Takushi Araya Dr. Daisuke Sasaki Dr. Atsuhiko Taniguchi Dr. Takeshi Sato Dr. Youhei Sohma Prof. Motomu Kanai 《Angewandte Chemie (International ed. in English)》2014,53(31):8236-8239
Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small‐molecule aggregation inhibitors of Alzheimer’s amyloid‐β (Aβ) are extremely scarce, however, and are mainly restricted to dye‐ and polyphenol‐type compounds that lack drug‐likeness. Based on the structure‐activity relationship of cyclic Aβ16–20 (cyclo‐[KLVFF]), we identified unique pharmacophore motifs comprising side‐chains of Leu2, Val3, Phe4, and Phe5 residues without involvement of the backbone amide bonds to inhibit Aβ aggregation. This finding allowed us to design non‐peptidic, small‐molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non‐peptidic, small‐molecule aggregation inhibitors of amyloids based on rational molecular design. 相似文献
9.
Abdul‐Hamid M. Emwas Zeyad A. Al‐Talla Xianrong Guo Suliman Al‐Ghamdi Harbi T. Al‐Masri 《Magnetic resonance in chemistry : MRC》2013,51(5):255-268
Copper is an essential nutrient for the normal development of the brain and nervous system, although the hallmark of several neurological diseases is a change in copper concentrations in the brain and central nervous system. Prion protein (PrP) is a copper‐binding, cell‐surface glycoprotein that exists in two alternatively folded conformations: a normal isoform (PrPC) and a disease‐associated isoform (PrPSc). Prion diseases are a group of lethal neurodegenerative disorders that develop as a result of conformational conversion of PrPC into PrPSc. The pathogenic mechanism that triggers this conformational transformation with the subsequent development of prion diseases remains unclear. It has, however, been shown repeatedly that copper plays a significant functional role in the conformational conversion of prion proteins. In this review, we focus on current research that seeks to clarify the conformational changes associated with prion diseases and the role of copper in this mechanism, with emphasis on the latest applications of NMR and EPR spectroscopy to probe the interactions of copper with prion proteins. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
10.
Isaak Beyer Dr. Nasrollah Rezaei‐Ghaleh Dr. Hans‐Wolfgang Klafki Dr. Olaf Jahn Dr. Ute Haußmann Prof. Dr. Jens Wiltfang Prof. Dr. Markus Zweckstetter Prof. Dr. Hans‐Joachim Knölker 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(25):8685-8693
In addition to the prototypic amyloid‐β (Aβ) peptides Aβ1–40 and Aβ1–42, several Aβ variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aβ variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid‐phase peptide synthesis of the N‐terminally elongated Aβ‐peptides Aβ?3–38, Aβ?3–40, and Aβ?3–42. Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aβ?3–38 and Aβ?3–40 are generated by transfected cells even in the presence of a tripartite β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aβ peptides starting at Val(?3) can be separated from N‐terminally‐truncated Aβ forms by high‐resolution isoelectric‐focusing techniques, despite virtually identical isoelectric points. The synthetic Aβ variants and the methods presented here are providing tools to advance our understanding of the potential roles of N‐terminally elongated Aβ variants in Alzheimer's disease. 相似文献
11.
Kunio Kimura Shin‐ichiro Kohama Satomi Kondoh Tetsuya Uchida Yuhiko Yamashita Takeshi Oohazama Yoshimitsu Sakaguchi 《Journal of polymer science. Part A, Polymer chemistry》2005,43(8):1624-1634
Polymerization of 4‐acetoxybenzoic acid (ABA) with 3,5‐diacetoxybenzoic acid (DABA) was examined to control the morphology of poly(p‐oxybenzoyl) (POB). Polymerizations were carried out at a concentration of 1.0% in an aromatic solvent Therm S‐1000® (mixture of dibenzyltoluene) at 320 °C. Polymerization of ABA yielded the POB fibrillar crystals, but the polymerization with DABA at a concentration in the feed (χf) of 0.10–0.15 afforded novel network structures comprised of spheres connected by fibrillar crystals. The diameter of the spheres prepared at χf of 0.15, which were 0.7 and 5.0 μm, showed bimodality. The network distance, fibril length, and fibril width were 6.1, 2.6, and 0.1 μm, respectively. They possessed high crystallinity. The network structure was formed as follows. Co‐oligomers were first precipitated in the beginning of the polymerization by liquid–liquid phase separation to form the microdroplets. The fibrillar crystals were formed in the coalesced spheres by the crystallization of oligomers induced by the increase of molecular weight. The fibrillar crystals connecting the spheres gradually appeared owing to the shrinkage of the spheres. The fibrillar crystals grew from the surface of the spheres with the crystallization of homo‐oligomers of 4‐oxybenzoyl units, and finally the network structure was completed. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1624–1634, 2005 相似文献
12.
Conformational switching of the prion protein into the abnormal form involves the formation of (obligatory) molten‐oligomers that mature into ordered amyloid fibrils. The role of water in directing the course of amyloid formation remains poorly understood. Here, we show that the mobility of the water molecules within the on‐pathway oligomers is highly retarded. The water relaxation time within the oligomers was estimated to be ≈1 ns which is about three orders of magnitude slower than the bulk water and resembles the characteristics of (trapped) nano‐confined water. We propose that the coalescence of these obligatory oligomers containing trapped water is entropically favored because of the release of ordered water molecules in the bulk milieu and results in the sequestration of favorable inter‐chain amyloid contacts via nucleated conformational conversion. The dynamic role of water in protein aggregation will have much broader implications in a variety of protein misfolding diseases. 相似文献
13.
Tatiana Pimenova Lukas Meier Bernd Roschitzki Gabriela Paraschiv Michael Przybylski Renato Zenobi 《Analytical and bioanalytical chemistry》2009,395(5):1395-1401
The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and
treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be
effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization
of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of
the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s).
In the epitope analysis of recombinant cellular bovine prion protein (bPrPC) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding
of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde
functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete
suppression of nonspecific bPrP binding. 相似文献
14.
Dr. Trung Quan Luong Nelli Erwin Matthias Neumann Andreas Schmidt Cornelia Loos Prof. Dr. Volker Schmidt Prof. Dr. Marcus Fändrich Prof. Dr. Roland Winter 《Angewandte Chemie (International ed. in English)》2016,55(40):12412-12416
We studied the combined effects of pressure (0.1–200 MPa) and temperature (22, 30, and 38 °C) on the catalytic activity of designed amyloid fibrils using a high‐pressure stopped‐flow system with rapid UV/Vis absorption detection. Complementary FT‐IR spectroscopic data revealed a remarkably high pressure and temperature stability of the fibrillar systems. High pressure enhances the esterase activity as a consequence of a negative activation volume at all temperatures (about ?14 cm3 mol?1). The enhancement is sustained in the whole temperature range covered, which allows a further acceleration of the enzymatic activity at high temperatures (activation energy 45–60 kJ mol?1). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils. 相似文献
15.
Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus
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Dr. Giuseppe Di Natale Dr. Ildikó Turi Dr. Giuseppe Pappalardo Prof. Imre Sóvágó Prof. Enrico Rizzarelli 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(10):4071-4084
Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrPC) to the disease‐related scrapie isoform (PrPSc). Copper(II) coordination to PrPC has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrPC. In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG11)3PrP(60‐114) [Ac=acetyl] as a model for the whole N‐terminus of the PrPC metal‐binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide‐bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide‐bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal‐binding‐affinity order observed in our previous studies. 相似文献
16.
《Chemistry & biology》1997,4(2):119-125
Background: Brain amyloid plaque, a diagnostic feature of Alzheimer's disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the β-amyloid protein (Aβ). As Aβ amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however.Results: Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Aβ variants Aβ1-40 and Aβ1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Aβ1-42 aggregates much more rapidly than Aβ1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Aβ amyloid protofibrils.Conclusions: Aβ protofibrils are likely to be intermediates in the in vitro assembly of Aβ amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Aβ aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Aβ protofibrils may be a useful therapeutic strategy. 相似文献
17.
Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on‐line solid‐phase extraction capillary electrophoresis‐mass spectrometry
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Lorena Ortiz‐Martin Fernando Benavente Silvia Medina‐Casanellas Estela Giménez Victoria Sanz‐Nebot 《Electrophoresis》2015,36(6):962-970
Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β‐protein (Aβ) (Aβ(1–15) and Aβ(10–20) peptides) by on‐line immobilized metal affinity SPE‐CE (IMA‐SPE‐CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25‐fold and 5‐fold decrease in the LODs by IMA‐SPE‐CE‐UV for Aβ(1–15) and Aβ(10–20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE‐UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA‐SPE‐CE‐MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10–20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10–20) peptide was good in a narrow concentration range (0.25–2.5 μg/mL, R2 = 0.93). Lastly, the potential of the optimized Ni(II)‐IMA‐SPE‐CE‐MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples. 相似文献
18.
Transfer of Copper from an Amyloid to a Natural Copper‐Carrier Peptide with a Specific Mediating Ligand
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Dr. Michel Nguyen Dr. Christian Bijani Nathalie Martins Prof. Dr. Bernard Meunier Dr. Anne Robert 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(47):17085-17090
The oxidative stress that arises from the catalytic reduction of dioxygen by CuII/I‐loaded amyloids is the major pathway for neuron death that occurs in Alzheimer’s disease. In this work, we show that bis‐8(aminoquinoline) ligands, copper(II) specific chelators, are able to catalytically extract CuII from Cu–Aβ1–16 and then completely release CuI in the presence of glutathione to provide a CuI–glutathione complex, a biological intermediate that is able to deliver copper to apo forms of copper–protein complexes. These data demonstrate that bis‐8(aminoquinolines) can perform the transfer of copper ions from the pathological Cu–amyloid complexes to regular copper–protein complexes. These copper‐specific ligands assist GSH to recycle CuI in an AD brain and consequently slow down oxidative damage that is due to copper dysregulation in Alzheimer’s disease. Under the same conditions, we have shown that the copper complex of PBT2, a mono(8‐hydroxyquinoline) previously used as a drug candidate, does not efficiently release copper in the presence of GSH. In addition, we report that GSH itself was unable to fully abstract copper ions from Cu–β‐amyloid complexes. 相似文献
19.
Tatsuya Ikenoue Prof. Dr. Young‐Ho Lee Prof. Dr. József Kardos Miyu Saiki Prof. Dr. Hisashi Yagi Prof. Dr. Yasushi Kawata Prof. Dr. Yuji Goto 《Angewandte Chemie (International ed. in English)》2014,53(30):7799-7804
Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores. 相似文献
20.
Aurélie Matéos Jean‐Michel Girardet Daniel Mollé Catherine Corbier Jean‐Luc Gaillard Laurent Miclo 《Rapid communications in mass spectrometry : RCM》2010,24(11):1533-1542
Equine β‐casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion‐exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro‐column. This method turned out to be an efficient tool to separate the CPPs Arg1–Lys34 and Glu4–Lys34 from non‐phosphorylated peptides. Purification was achieved by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and each CPP was hydrolyzed by endoproteinase Glu‐C. Finally, the digests were analyzed by RP‐HPLC/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS) and identified by nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) to locate the phosphorylated sites of the β‐casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser9, Ser23, Ser24, and Ser25. Addition of phosphate groups on Ser18, Thr12, and Ser10 led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine β‐casein follows a sequential way and is not randomly performed. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献