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1.
Dr. Yasuhiro Kajihara Yasutaka Tanabe Shun Sasaoka Dr. Ryo Okamoto 《Chemistry (Weinheim an der Bergstrasse, Germany)》2012,18(19):5944-5953
The N‐glycosylation of proteins is generated at the consensus sequence NXS/T (where X is any amino acid except proline) by the biosynthetic process, and occurs in the endoplasmic reticulum and Golgi apparatus. In order to investigate the influence of human complex‐type oligosaccharides on counterpart protein conformation, crambin and ovomucoide, which consist of 46 and 56 amino acid residues, respectively, were selected for synthesis of model glycoproteins. These small glycoproteins were intentionally designed to be glycosylated at the α‐helix (crambin: 8 position), β‐sheet (crambin: 2 position) and loop position between the antiparallel β‐sheets (ovomucoide: 28 position), and were synthesized by using a peptide‐segment coupling strategy. After preparation of these glycosylated polypeptide chains, protein folding experiments were performed under redox conditions by using cysteine–cystine. Although the small glycoproteins bearing intentional glycosylation at the α‐helix and β‐sheet exhibited a suitable folding process, glycosylation at the loop position between the antiparallel β‐strands caused multiple products. The conformational differences in the isolated homogeneous glycoproteins compared with non‐glycosylated counterparts were evaluated by circular dichroism (CD) and NMR spectroscopy. These analyses suggested that this intentional N‐glycosylation did not result in large conformational changes in the purified protein structures, including the case of glycosylation at the loop position between the antiparallel β‐strands. In addition to these experiments, the conformational properties of three glycoproteins were evaluated by CD spectroscopy under different temperatures. The oligosaccharides on the protein surface fluctuated considerably; this was dependent on the increase in the solution temperature and was thought to disrupt the protein tertiary structure. Based on the measurement of the CD spectra, however, the glycoproteins bearing three disulfide bonds did not exhibit any change in their protein tertiary structure. These results suggest that the oligosaccharide conformational fluctuations were not disruptive to protein tertiary structure, and the tertiary structure of glycoproteins might be stabilized by the disulfide bond network. 相似文献
2.
Synthesis of Glc1Man9‐Glycoprotein Probes by a Misfolding/Enzymatic Glucosylation/Misfolding Sequence 下载免费PDF全文
Dr. Masayuki Izumi Yukiho Oka Dr. Ryo Okamoto Dr. Akira Seko Dr. Yoichi Takeda Prof. Dr. Yukishige Ito Prof. Dr. Yasuhiro Kajihara 《Angewandte Chemie (International ed. in English)》2016,55(12):3968-3971
Glycoproteins in non‐native conformations are often toxic to cells and may cause diseases, thus the quality control (QC) system eliminates these unwanted species. Lectin chaperone calreticulin and glucosidase II, both of which recognize the Glc1Man9 oligosaccharide on glycoproteins, are important components of the glycoprotein QC system. Reported herein is the preparation of Glc1Man9‐glycoproteins in both native and non‐native conformations by using the following sequence: misfolding of chemically synthesized Man9‐glycoprotein, enzymatic glucosylation, and another misfolding step. By using synthetic glycoprotein probes, calreticulin was found to bind preferentially to a hydrophobic non‐native glycoprotein whereas glucosidase II activity was not affected by glycoprotein conformation. The results demonstrate the ability of chemical synthesis to deliver homogeneous glycoproteins in several non‐native conformations for probing the glycoprotein QC system. 相似文献
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Christian Piontek Dr. Daniel Varón Silva Dr. Christian Heinlein Claudia Pöhner Stefano Mezzato Dr. Petra Ring Andreas Martin Dr. Franz Xaver Schmid Prof. Carlo Unverzagt Prof. 《Angewandte Chemie (International ed. in English)》2009,48(11):1941-1945
Active RNase glycoprotein from three pieces : The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N‐glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.
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Christian Piontek Dr. Petra Ring Olaf Harjes Christian Heinlein Stefano Mezzato Dr. Nelson Lombana Claudia Pöhner Markus Püttner Dr. Daniel Varón Silva Dr. Andreas Martin Dr. Franz Xaver Schmid Prof. Carlo Unverzagt Prof. 《Angewandte Chemie (International ed. in English)》2009,48(11):1936-1940
Seven in one blow : The efficient formation of mixed disulfides on the thiol‐rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.
6.
Mag. Moritz Schennach Priv.‐Doz. Dr. Kathrin Breuker 《Angewandte Chemie (International ed. in English)》2014,53(1):164-168
Proteins can be exposed to vastly different environments such as the cytosol or membranes, but the delicate balance between external factors and intrinsic determinants of protein structure, stability, and folding is only poorly understood. Here we used electron capture dissociation to study horse and tuna heart Cytochromes c in the complete absence of solvent. The significantly different stability of their highly similar native folds after transfer into the gas phase, and their strikingly different folding behavior in the gas phase, can be rationalized on the basis of electrostatic interactions such as salt bridges. In the absence of hydrophobic bonding, protein folding is far slower and more complex than in solution. 相似文献
7.
Lijuan Zhang Yawei Xu Hailiang Yao Liqi Xie Jun Yao Haojie Lu Prof. Pengyuan Yang Prof. 《Chemistry (Weinheim an der Bergstrasse, Germany)》2009,15(39):10158-10166
A core–satellite‐structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. This novel hybrid material is composed of a silica‐coated ferrite “core” and numerous “satellites” of gold nanoparticles with lots of “anchors”. The anchor, 3‐aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap‐and‐release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6 % separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79 mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N‐glycosylation sites. In all, 194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1 %) were newly identified. 相似文献
8.
Dr. Thomas Raschle Perla Rios Flores Christian Opitz Prof. Dr. Daniel J. Müller Prof. Dr. Sebastian Hiller 《Angewandte Chemie (International ed. in English)》2016,55(20):5952-5955
β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale. 相似文献
9.
Boronate‐Affinity Glycan‐Oriented Surface Imprinting: A New Strategy to Mimic Lectins for the Recognition of an Intact Glycoprotein and Its Characteristic Fragments 下载免费PDF全文
Zijun Bie Yang Chen Jin Ye Shuangshou Wang Prof. Dr. Zhen Liu 《Angewandte Chemie (International ed. in English)》2015,54(35):10211-10215
Lectins possess unique binding properties and are of particular value in molecular recognition. However, lectins suffer from several disadvantages, such as being hard to prepare and showing poor storage stability. Boronate‐affinity glycan‐oriented surface imprinting was developed as a new strategy for the preparation of lectin‐like molecularly imprinted polymers (MIPs). The prepared MIPs could specifically recognize an intact glycoprotein and its characteristic fragments, even within a complex sample matrix. Glycan‐imprinted MIPs could thus prove to be powerful tools for important applications such as proteomics, glycomics, and diagnostics. 相似文献
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Coordination‐Driven Folding and Assembly of a Short Peptide into a Protein‐like Two‐Nanometer‐Sized Channel 下载免费PDF全文
Dr. Tomohisa Sawada Asami Matsumoto Prof. Dr. Makoto Fujita 《Angewandte Chemie (International ed. in English)》2014,53(28):7228-7232
Short peptide helices have attracted attention as suitable building blocks for soft functional materials, but they are rarely seen in crystalline materials. A new artificial nanoassembly of short peptide helices in the crystalline state is presented in which peptide helices are arranged three‐dimensionally by metal coordination. The folding and assembly processes of a short peptide ligand containing the Gly‐Pro‐Pro sequence were induced by silver(I) coordination in aqueous alcohol, and gave rise to a single crystal composed of polyproline II helices. Crystallographic studies revealed that this material possesses two types of unique helical nanochannel; the larger channel measures more than 2 nm in diameter. Guest uptake properties were investigated by soaking the crystals in polar solutions of guest molecules; anions, organic chiral molecules, and bio‐oligomers are effectively encapsulated by this peptide‐folded porous crystal, with moderate to high chiral recognition for chiral molecules. 相似文献
11.
Dr. Nitin A. Patil Dr. John A. Karas Prof. Dr. John D. Wade Dr. Mohammed Akhter Hossain Dr. Julien Tailhades 《Chemistry (Weinheim an der Bergstrasse, Germany)》2019,25(36):8599-8603
Structure–activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated “one-pot” strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin. 相似文献
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Prof. Yukishige Ito Prof. Yasuhiro Kajihara Prof. Yoichi Takeda 《Chemistry (Weinheim an der Bergstrasse, Germany)》2020,26(67):15461-15470
The introduction of Asn-linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin-chaperones calnexin (CNX) and calreticulin (CRT), glucosidase II (G-II), and UDP-Glc:glycoprotein glucosyltransferase (UGGT). G-II initiates glycoproteins’ entry and exit from the cycle, and UGGT serves as the “folding sensor”. This account summarizes our effort to analyze the properties of enzymes and lectins that play important roles in GPQC, especially those involved in the CNX/CRT cycle. To commence our study, general methods for the synthesis of high-mannose-type glycans and glycoproteins were established. Based on these, various substrates to analyze components of the GPQC were created, and properties of CRT, G-II, and UGGT have been clarified. 相似文献
14.
Minna Kortelainen Aku Suhonen Dr. Andrea Hamza Dr. Imre Pápai Dr. Elisa Nauha Dr. Sanna Yliniemelä‐Sipari Prof. Dr. Maija Nissinen Prof. Dr. Petri M. Pihko 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(26):9493-9504
A series of small, unsymmetrical pyridine‐2,6‐dicarboxylamide oligoamide foldamers with varying lengths and substituents at the end groups were synthetized to study their conformational properties and folding patterns. The @‐type folding pattern resembled the oxyanion‐hole motifs of enzymes, but several alternative folding patterns could also be characterized. Computational studies revealed several alternative conformers of nearly equal stability. These folding patterns differed from each other in their intramolecular hydrogen‐bonding patterns and aryl–aryl interactions. In the solid state, the foldamers adopted either the globular @‐type fold or the more extended S‐type conformers, which were very similar to those foldamers obtained computationally. In some cases, the same foldamer molecule could even crystallize into two different folding patterns, thus confirming that the different folding patterns are very close in energy in spite of their completely different shapes. Finally, the best match for the observed NOE interactions in the liquid state was a conformation that matched the computationally characterized helix‐type fold. 相似文献
15.
Beynon RJ 《Journal of mass spectrometry : JMS》2004,39(2):188-192
In a recent paper, Villaneuva et al. (J. Mass Spectrom. 2002; 37: 974) described the use of exoproteases as probes of higher order structure in proteins. Their model assumes that the proteins are attacked sequentially from either the N-terminus or the C-terminus, depending on the type of exoprotease (aminopeptidase or carboxypeptidase) used. The products of this presumed exoproteolysis were then analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The pattern of fragments obtained was mapped on to the primary sequence of the protein, and the exoproteolysis was interpreted as comprising a series of fast and slow phases, the rates of the different phases being directly related to the higher order structure of particular segments of the protein. Here, it is shown that this explanation is unlikely, that both kinetic and practical considerations suggest that alternative explanations for the data should be sought, and that exoproteolysis will perhaps not be as valuable as a conformational probe as the authors suggest. 相似文献
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Dr. Eva Di Silvio Prof. Maurizio Brunori Prof. Stefano Gianni 《Angewandte Chemie (International ed. in English)》2015,54(37):10867-10869
The funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site‐directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy‐landscape theory. 相似文献
18.
Dr. Jozef Adamcik Prof. Dr. Raffaele Mezzenga 《Angewandte Chemie (International ed. in English)》2018,57(28):8370-8382
Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on‐ or off‐pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon?crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape. 相似文献
19.
Atsushi Suenaga Dr. Tetsu Narumi Dr. Noriyuki Futatsugi Dr. Ryoko Yanai Yousuke Ohno Dr. Noriaki Okimoto Dr. Makoto Taiji Dr. 《化学:亚洲杂志》2007,2(5):591-598
Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa. 相似文献
20.
Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study 下载免费PDF全文
Dr. Jongcheol Seo Waldemar Hoffmann Dr. Stephan Warnke Prof. Dr. Michael T. Bowers Prof. Dr. Kevin Pagel Dr. Gert von Helden 《Angewandte Chemie (International ed. in English)》2016,55(45):14173-14176
Can the structures of small to medium‐sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion‐mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β‐lactoglobulin, which are prototypical examples of helical and β‐sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved. 相似文献