Content
2011
Optochemical Genetics
16.12.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201103236,
Volume 50, Issue 51, pages 12156–12182,
published on 16.12.2011
Photochromic channel blockers provide a conceptually simple and
convenient way to modulate neuronal activity with light. We have recently described a family of azobenzenes that function as tonic blockers of Kv channels but require UV-A light to unblock and need to be actively switched by toggling between two different wavelengths. We now introduce red-shifted compounds that fully operate in the visible region of the spectrum and quickly turn themselves off in the dark. Furthermore, we have developed a version that does not block
effectively in the dark-adapted state, can be switched to a blocking state with blue light, and reverts to the inactive state automatically. Photochromic blockers of this type could be useful for the photopharmacological control of neuronal activity under mild conditions.
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Improved Synthesis and Mutagenicity of Oligonucleotides Containing 5-Hydroxymethylcytosine, 5-Formylcytosine and 5-Carboxylcytosine
02.12.2011
Chemistry,
2011,
DOI: 10.1002/chem.201102782,
Volume 17, Issue 49, pages 13782–13788
published on 02.12.2011
5-Formylcytosine (fC or 5-CHOdC) and 5-carboxylcytosine (caC or 5-COOHdC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or 5-MedC) via 5-hydroxymethylcytosine (hmC or 5-HOMedC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd0-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.
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Vibralactone as a Tool to Study the Activity and Structure of the ClpP1P2 Complex from Listeria monocytogenes
11.11.2011
Angewandte Chemie,
2011,
DOI: 10.1002/ange.201104391,
Volume 50, Issue 46, pages 11001–11004
published on 11.11.2011
Nature provides a rich source of bioactive compounds comprising a diverse set of electrophilic core structures that are poised to react with corresponding nucleophilic residues such as serine and cysteine in enzyme active sites.These residues are usually relevant for catalysis and therefore display fine-tuned reactivity towards their dedicated substrates.We and others previously investigated the dedicated targets of monocyclic b-lactones which turned out to be
potent and selective inhibitors of diverse disease-associated enzyme classes. Covalent inhibition of the caseinolytic peptidase ClpP, for instance, resulted in a dramatic attenuation of bacterial virulence. ClpP is an important, highly conserved heat shock protein with additional regulatory functions in many pathogens. Some organisms such as Listeria monocytogenes genetically encode for two functionally and structurally uncharacterized ClpP isoforms (ClpP1 and ClpP2). So far, all b-lactones were reported to target
solely ClpP2 and not ClpP1, raising the question whether monocyclic lactones lack suitable reactivity to interact with the ClpP1 active-site nucleophile.
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Deep proteome and transcriptome mapping of a human cancer cell line
08.11.2011
Molecular Systems Biology,
2011,
doi:10.1038/msb.2011.81,
7 Article number: 548
published on 08.11.2011
Molecular Systems Biology, online article
Molecular Systems Biology, online article
While the number and identity of proteins expressed in a single human cell type is currently unknown, this fundamental question can be addressed by advanced mass spectrometry (MS)-based proteomics. Online liquid chromatography coupled to high-resolution MS and MS/MS yielded 166 420 peptides with unique amino-acid sequence from HeLa cells. These peptides identified 10 255 different human proteins encoded by 9207 human genes, providing a lower limit on the proteome in this cancer cell line. Deep transcriptome sequencing revealed transcripts for nearly all detected proteins. We calculate copy numbers for the expressed proteins and show that the abundances of >90% of them are within a factor 60 of the median protein expression level. Comparisons of the proteome and the transcriptome, and analysis of protein complex databases and GO categories, suggest that we achieved deep coverage of the functional transcriptome and the proteome of a single cell type.
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The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling
07.10.2011
EMBO reports,
2011,
doi:10.1038/embor.2011.190,
12, 1127 - 1134
published on 07.10.2011
RIG-I detects cytosolic viral dsRNA with 5′ triphosphates (5′-ppp-dsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a ‘C’-shaped structure. Domains 1A and 2A are maintained in a ‘signal-off’ state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5′-ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA- and ATP-driven structural switch in the SF2 domain.
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Systems-Based Analysis of Modified tRNA Bases
04.10.2011
Angewandte Chemie,
2011,
DOI: 10.1002/ange.201103229,
Volume 123, Issue 41, pages 9913–9916
published on 04.10.2011
The genetic system contains several levels of information. Firstly, the sequence of the canonical bases A, C, G, and T/U in DNA and RNA encodes amino acids through specific base triplets. Secondly, the methylation status of the cytosine base in DNA imprints epigenetic information into the genetic system, thereby contributing to the division of genes into active and inactive elements. Both information layers are chemically well investigated. Less is known about a putative third level of information associated with the chemical
modification of RNA nucleobases. Although RNA, and in particular tRNA, is known to contain more than 100 different modified nucleosides, the exact type of information added by base modification is largely unknown. A number of common modifications have been shown to improve the maintenance of the reading frame, influence RNA stability, and to be involved in proof-reading by tRNA synthetases.
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Pictures of the Scientific Oktoberfest 2011
22.09.2011
2011,
published on 22.09.2011
Thanks to everyone of the 360 people who attended the „Scientific Oktoberfest 2011“! We were able to experience amazing science combined with a awesome spirit.
Thank you!
Here you can see some impressions.
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Scientific Oktoberfest: CIPSM-Fest of Chemical Biology 2011
15.09.2011
The „Scientific Oktoberfest“ will be this years meeting point for some of the internationally most distinguished researchers in the field of Chemical Biology. The so-called „CIPSM Fest of Chemical Biology“ will be held from the 15th to the 16th of September in the Department of Chemistry of the Ludwig-Maximilians-Universität (LMU) München.
At the two-day conference, 400 researchers from around the world will discuss state-of-the art research in various fields spanning from chemistry to biology and present their latest results in the fight against diseases like cancer or bacterial infections.
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To register click here to get to the webpage of the CIPSM-Fest of Biological Chemistry 2011
Program of the Scientific Oktoberfest
Press release of the Scientific Oktoberfest
Pressemitteilung für das Wissenschaftliche Oktoberfest
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Design, Synthesis, and Functionalization of Dimeric Peptides Targeting Chemokine Receptor CXCR4
12.09.2011
J. Med. Chem.,
2011,
DOI: 10.1021/jm2009716,
54 (21), pp 7648–7662
published on 12.09.2011
The chemokine receptor CXCR4 is a critical regulator of inflammation and immune surveillance, and it is specifically implicated in cancer metastasis and HIV-1 infection. On the basis of the observation that several of the known antagonists remarkably share a C2 symmetry element, we constructed symmetric dimers with excellent antagonistic activity using a derivative of a cyclic pentapeptide as monomer. To optimize the binding affinity, we investigated the influence of the distance between the monomers and the pharmacophoric sites in the synthesized constructs. The affinity studies in combination with docking computations support a two-site binding model. In a final step, 1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was introduced as chelator for (radio-)metals, thus allowing to exploit these compounds as a new group of CXCR4-binding peptidic probes for molecular imaging and endoradiotherapeutic purposes. Both the DOTA conjugates and some of their corresponding metal complexes retain good CXCR4 affinity, and one 68Ga labeled compound was studied as PET tracer.
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Reversible bond formation enables the replication and amplification of a crosslinking salen complex as an orthogonal base pair
28.08.2011
Nature Chemistry,
2011,
doi:10.1038/nchem.1117,
3, 794–800
published on 28.08.2011
The universal genetic code relies on two hydrogen-bonded Watson–Crick base pairs that can form 64 triplet codons. This places a limit on the number of amino acids that can be encoded, which has motivated efforts to create synthetic base pairs that are orthogonal to the natural ones. An additional base pair would result in another 61 triplet codons. Artificial organic base pairs have been described in enzymatic incorporation studies, and inorganic T–Hg–T and C–Ag–C base pairs have been reported to form in primer extension studies. Here, we demonstrate a metal base pair that is fully orthogonal and can be replicated, and can even be amplified by polymerase chain reaction in the presence of the canonical pairs dA:dT and dG:dC. Crystal structures of a dS–Cu–dS base pair inside a polymerase show that reversible chemistry is possible directly inside the polymerase, which enables the efficient copying of the inorganic crosslink. The results open up the possibility of replicating and amplifying artificial inorganic DNA nanostructures by extending the genetic alphabet.
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The Discovery of 5-Formylcytosine in Embryonic Stem Cell DNA
25.07.2011
Angewandte Chemie,
2011,
DOI: 10.1002/ange.201103899,
Volume 123, Issue 31, pages 7146–7150
published on 25.07.2011
Cellular development requires the silencing and activation of
specific gene sequences in a well-orchestrated fashion. Transcriptional gene silencing is associated with the clustered
methylation of cytosine bases (C) in CpG units of promoters.
The methylation occurs at position C5 of cytosine to give 5-
methylcytosine (mC) with the help of special DNA methyltransferases (DNMT). The DNA methylome is significantly
reprogrammed at various stages during early development,
during the development of primordial germ cells, or later
in a locus-specific way at postdevelopmental stages. Decreasing levels of mC can be established passively by
successive rounds of DNA replication in the absence of
methyltransferases. Active demethylation, in contrast, is
proposed to be a process in which the mC bases are directly
converted back into unmodified cytosines in the genome. The recent discovery that mC can be further oxidized to hydroxymethylcytosine (hmC) with the help of TET enzymes has led to the idea that hmC is connected to epigenetic reprogramming, maybe as an intermediate in an, as yet controversial, active demethylation process. Indeed recent data suggest that active demethylation in postdevelopmental phases may proceed through deamination of hmC to give 5-hydroxymethyluridine (hmU), which is then removed from the genome with the help of the base excision repair (BER) system. Chemically, an attractive alternative mechanism for a more global active demethylation could be envisioned through further oxidation of hmC to give either 5- formylcytosine (fC) or 5-carboxylcytosine (caC) followed by elimination of a formyl or carboxyl group, respectively . Although such an oxidative active demethylation pathway with hmC as the starting point has been
frequently postulated, none of the further oxidized bases
(fC, caC) have so far been detected.
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PET Imaging of CXCR4 Receptors in Cancer by a New Optimized Ligand
20.07.2011
ChemMedChem,
2011,
DOI: 10.1002/cmdc.201100320,
Volume 6, Issue 10, pages 1789–1791
published on 20.07.2011
Nowadays, personalized medicine is considered to be of utmost importance to target the different causes of identical phenotypes. For example, cancer of the same type can significantly differ in its biochemical phenotypes and thus its molecular profile between patients. The disease-specific characterization of malignant cells at the molecular level is a prerequisite for targeted therapy and personalized treatment. Positron emission tomography (PET) and its combination with computer tomography (PET/CT) and magnetic resonance tomography
(PET/MRT) in modern hybrid systems offer the possibility to localize and quantify biochemical function by means of PET with
anatomical (CT) and morphological (MRT) information. For this
purpose, radiolabeled probes are used that target, for example,
enzyme activities, transport systems, and surface receptors with high affinity and specificity. We describe the development of the first gallium-68 (t1/2=68 min) ligand for the G-protein- coupled receptor CXCR4 and preliminary demonstrate its potential for in vivo imaging of CXCR4 expression using a mouse model with a human small-cell lung cancer xenograft. This ligand offers the possibility to be used as an initial tool for diagnosis in an approach of personalized medicine for treating CXCR4-related cancer.
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5-Hydroxymethylcytosine, the Sixth Base of the Genome
11.07.2011
Angewandte Chemie,
2011,
DOI: 10.1002/ange.201101547,
Volume 123, Issue 29, pages 6588–6596
published on 11.07.2011
5-Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5-methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection—most notably LC-MS and glucosyltransferase assays-helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue-specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis.
Evidence is currently accumulating that hmC may not exclusively
be an intermediate of an active demethylation process, but that it
functions instead as an important epigenetic marker.
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Unraveling the Protein Targets of Vancomycin in Living S. aureus and E. faecalis Cells
07.07.2011
Jacs,
2011,
DOI: 10.1021/ja2039979,
133 (31), pp 12144–12153
published on 07.07.2011
Vancomycin is a potent glycopeptide antibiotic that has evolved to specifically bind to the D-Ala-D-Ala dipeptide termini of nascent peptidoglycans. Although this mode of action is well established, several studies indicate that vancomycin and analogues exploit noncanonical target sites. In order to address all vancomycin targets in clinically relevant Staphylococcus aureus and Enterococcus faecalis strains we developed a series of small-molecule photoaffinity probes based on vancomycin. Proteomic profiling revealed the specific labeling of two previously unknown vancomycin
targets that are likely to contribute to its antibiotic activity. The specific inhibition of the major staphylococcal autolysin Atl confirms previous observations that vancomycin alters S. aureus cell morphology by interaction with the autolytic machinery. Moreover, in E. faecalis the vancomycin photoprobe specifically binds to an ABC transporter protein, which likely impedes the uptake of essential nutrients such as sugars and peptides. The labeling of these two prominent membrane targets in living cells reveals a thus far unexplored mode of vancomycin binding and inhibition that could allow a rational design of variants with improved activity.
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Recognition of 5-Hydroxymethylcytosine by the Uhrf1 SRA Domain
22.06.2011
PLoS ONE,
2011,
doi:10.1371/journal.pone.0021306,
6(6): e21306.
published on 22.06.2011
PLoS ONE, online article
PLoS ONE, online article
Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group.
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An efficient synthesis of loline alkaloids
19.06.2011
Nature Chemistry,
2011,
doi:10.1038/nchem.1072,
3, 543–545
published on 19.06.2011
Nature Chemistry, online article
Nature Chemistry, online article
Loline is a small alkaloid that, in spite of its simple-looking structure, has posed surprising challenges to synthetic chemists. It has been known for more than a century and has been the subject of extensive biological investigations, but only two total syntheses have been achieved to date. Here, we report an asymmetric total synthesis of loline that, with less then ten steps, is remarkably short. Our synthesis incorporates a Sharpless epoxidation, a Grubbs olefin metathesis and an unprecedented transannular aminobromination, which converts an eight-membered cyclic carbamate into a bromopyrrolizidine. The synthesis is marked by a high degree of chemo- and stereoselectivity and gives access to several members of the loline alkaloid family. It delivers sufficient material to support a programme aimed at studying the complex interactions between plants, fungi, insects and bacteria brokered by loline alkaloids.
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Tuning Photochromic Ion Channel Blockers
09.06.2011
ACS Chem. Neuroscience,
2011,
DOI: 10.1021/cn200037p,
2 (9), pp 536–543
published on 09.06.2011
Photochromic channel blockers provide a conceptually simple and
convenient way to modulate neuronal activity with light. We have recently described a family of azobenzenes that function as tonic blockers of Kv channels but require UV-A light to unblock and need to be actively switched by toggling between two different wavelengths. We now introduce red-shifted compounds that fully operate in the visible region of the spectrum and quickly turn themselves off in the dark. Furthermore, we have developed a version that does not block
effectively in the dark-adapted state, can be switched to a blocking state with blue light, and reverts to the inactive state automatically. Photochromic blockers of this type could be useful for the photopharmacological control of neuronal activity under mild conditions.
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Identification and characterization of a novel ubiquitous nucleolar protein ‘NARR’ encoded by a gene overlapping the rab34 oncogene
17.05.2011
Nucl. Acids Res.,
2011,
doi: 10.1093/nar/gkr273,
published on 17.05.2011
Nucl. Acids Res., online article
Nucl. Acids Res., online article
There are only few reports on protein products originating from overlapping mammalian genes even though computational predictions suggest that an appreciable fraction of mammalian genes could potentially overlap. Mass spectrometry-based
proteomics has now acquired the tools to probe proteins in an unbiased manner, providing direct evidence of the output of the genomic and gene expression machinery. In particular, proteomics can refine gene predictions and discover novel geneprocessing events and gene arrangements. Here, we report the mass spectrometric discovery and biochemical validation of the novel protein encoded by a gene overlapping rab34 oncogene. The novel protein is highly conserved in mammals. In humans, it contains 13 distinct Nine-Amino acid
Residue-Repeats (NARR) with the consensus sequence PRVIV(S/T)PR in which the serine or threonine residues are phosphorylated during M-phase. NARR is ubiquitously expressed and resides in nucleoli where it colocalizes with ribosomal DNA (rDNA) gene clusters. Its distribution only
partially overlaps with upstream binding factor, one of the main regulators of RNA Polymerase I activity, and is entirely uncoupled from it in mitotic cells and upon inhibition of transcription. NARR only partially colocalizes with fibrillarin, the pre-ribosomal
RNA-processing protein, positioning NARR in a separate niche within the rDNA cluster.
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A Conformational Switch Underlies ClpP Protease Function
04.05.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201100666,
Volume 50, Issue 25, pages 5749–5752
published on 04.05.2011
The barrel-shaped serine protease ClpP degrades misfolded, damaged, and regulatory proteins. Substrate proteins enter the ClpP barrel through the two axial pores, but it is unclear how the peptide products exit the barrel. Here we report the structure of ClpP from Staphylococcus aureus, which reveals a previously unobserved compressed state of the barrel. A conformational switch in the active center “handle region” results in closure of the active sites and opening of equatorial pores. Conserved residues in the handle region underlie the conformational switch and are functionally essential although they are not part of the active sites. These results are consistent with processive cycling of ClpP between an extended state with open active sites and closed equatorial pores, and a compressed state with closed active sites and open pores for product release.
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High Recovery FASP Applied to the Proteomic Analysis of Microdissected Formalin Fixed Paraffin Embedded Cancer Tissues Retrieves Known Colon Cancer Markers
28.04.2011
J. Proteome Res,
2011,
DOI: 10.1021/pr200019m,
published on 28.04.2011
J. Proteome Res, online article
J. Proteome Res, online article
Proteomic analysis of samples isolated by laser capture microdissection from clinical specimens requires sample preparation and fractionation methods suitable for small amounts of protein. Here we describe a streamlined filter-aided sample preparation (FASP) workflow that allows efficient analysis of lysates from low numbers of cells. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields in the low to submicrogram range. In a single LC–MS/MS run, analyses of 500, 1000, and 3000 cells allowed identification of 905, 1536, and 2055 proteins, respectively. Incorporation of an additional SAX fractionation step at somewhat higher amounts enabled the analysis of formalin fixed and paraffin embedded human tissues prepared by LCM to a depth of 3600–4400 proteins per single experiment. We applied this workflow to compare archival neoplastic and matched normal colonic mucosa cancer specimens for three patients. Label-free quantification of more than 6000 proteins verified this technology through the differential expression of 30 known colon cancer markers. These included Carcino-Embryonic Antigen (CEA), the most widely used colon cancer marker, complement decay accelerating factor (DAF, CD55) and Metastasis-associated in colon cancer protein 1 (MACC1). Concordant with literature knowledge, mucin 1 was overexpressed and mucin 2 underexpressed in all three patients. These results show that FASP is suitable for the low level analysis of microdissected tissue and that it has the potential for exploration of clinical samples for biomarker and drug target discovery.
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A Novel Two-Stage Tandem Mass Spectrometry Approach and Scoring Scheme for the Identification of O-GlcNAc Modified Peptides
26.03.2011
American Society for Mass Spectrometry,
2011,
DOI: 10.1007/s13361-011-0107-y,
Volume 22, Number 5, 931-942
published on 26.03.2011
American Society for Mass Spectrometry, online article
American Society for Mass Spectrometry, online article
The modification of serine and threonine residues in proteins by a single N-acetylglucosamine (O-GlcNAc) residue is an emerging post-translational modification (PTM) with broad biological implications. However, the systematic or large-scale analysis of this PTM is hampered by several factors, including low stoichiometry and the lability of the O-glycosidic bond during tandem mass spectrometry. Using a library of 72 synthetic glycopeptides, we developed a two-stage tandem MS approach consisting of pulsed Q dissociation (PQD) for O-GlcNAc peptide detection and electron transfer dissociation (ETD) for identification and site localization. Based on a set of O-GlcNAc
specific fragment ions, we further developed a score (OScore) that discriminates O-GlcNAc peptide spectra from spectra of unmodified peptides with 95% sensitivity and 999% specificity. Integrating the OScore into the two-stage LC-MS/MS approach detected O-GlcNAc peptides in the low fmol range and at 10-fold better sensitivity than a single data-dependent ETD tandem MS experiment.
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Discovery and Synthesis of New UV-Induced Intrastrand C(4−8)G and G(8−4)C Photolesions
22.03.2011
J. Am. Chem. Soc.,
2011,
DOI: 10.1021/ja111304f,
133 (14), pp 5186–5189
published on 22.03.2011
UV irradiation of cellular DNA leads to the formation of a number of defined mutagenic DNA lesions. Here we report the discovery of new intrastrand C(4−8)G and G(8−4)C cross-link lesions in which the C(4) amino group of the cytosine base is covalently linked to the C(8) position of an adjacent dG base. The structure of the novel lesions was clarified by HPLC−MS/MS data for UV-irradiated DNA in combination with chemical synthesis and direct comparison of the synthetic material with irradiated DNA. We also report the ability to generate the lesions directly in DNA with the help of a photoactive precursor that was site-specifically incorporated into DNA. This should enable detailed chemical and biochemical investigations of these lesions.
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Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases
12.03.2011
Bioorganic & Medicinal Chemistry,
2011,
doi:10.1016/j.bmc.2011.03.014,
published on 12.03.2011
Bioorganic & Medicinal Chemistry, online article
Bioorganic & Medicinal Chemistry, online article
Activity-based probes (ABPs) have found increasing use in functional proteomics studies. Recently, ABPs that can be employed in combination with click chemistry gained particular attention due to their flexible application in vitro and in vivo. Moreover, there is a continuous need for new ABPs that target small subsets of enzymes. We here report novel clickable ABPs based on the 4-chloro-isocoumarin (IC) electrophile, a mechanism-based inhibitor scaffold that covalently binds serine proteases. We describe the synthesis of a small library of IC ABPs containing an alkyne function and a set of diverse selectivity elements. The different substituents on the IC structure determine which proteases are bound, showing good correlation with the preferred substrate preferences. The IC ABPs can detect their target proteases in a proteome background in a sensitive manner (down to 0.007% of total protein). Furthermore, we show activity-dependent and selective labeling of endogenous proteases in a tissue proteome. These ICs therefore represent a valuable extension to already existing ABPs for serine proteases and may be instrumental in future elucidation of serine protease functions.
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Structure and dynamics of the lipid modifications of a transmembrane Alpha-helical peptide determined by 2H solid-state NMR spectroscopy
03.03.2011
Biochimica et Biophysica Acta,
2011,
doi:10.1016/j.bbamem.2010.12.015,
Volume 1808, Issue 3, March 2011, Pages 784-791
published on 03.03.2011
Biochimica et Biophysica Acta, online article
Biochimica et Biophysica Acta, online article
The fusion of biological membranes is mediated by integral membrane proteins with Alpha-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed Alpha-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the Nterminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the 2H and 31P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order
parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant.
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Biosynthesis of a Fluorescent Protein with Extreme Pseudo-Stokes Shift by Introducing a Genetically Encoded Non-Natural Amino Acid outside the Fluorophore
22.02.2011
A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifically incorporated into the recombinant enhanced cyan fluorescent protein (eCFP) at a permissible surface position ∼20 Å away from the protein fluorophore using amber suppression in Escherichia coli with an engineered cognate Methanococcus jannaschii tRNA synthetase. The resulting eCFPCou exhibited almost quantitative intramolecular Förster resonance energy transfer (FRET) between its two fluorophores, showing brilliant cyan emission at 476 nm upon excitation in the near-UV at 365 nm (a wavelength easily accessible via conventional laboratory UV sources), in contrast to its natural counterpart. Thus, this fluorescent protein with unprecedented spectroscopic properties reveals an extreme apparent Stokes shift of ∼110 nm between the absorption wavelength of the coumaryl group and the emission wavelength of eCFP.
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Synthetic Studies toward A-74528
16.02.2011
Organic Letters,
2011,
DOI: 10.1021/ol103117j,
13 (6), pp 1386–1389
published on 16.02.2011
A potentially biomimetic approach toward the complex polyketide A-74528 is described. It is based on highly substituted biaryl compounds, synthesized using advanced cross-coupling and condensation methodologies.
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Concise Total Syntheses of Variecolortides A and B through an Unusual Hetero-Diels–Alder Reaction
07.02.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201006154,
Volume 50, Issue 6, pages 1402–1405
published on 07.02.2011
Speculations on the biosynthetic origin of natural products continue to inspire synthetic chemists and have led to many elegant and efficient total syntheses. Many of these involve cascade reactions, where a high-energy substrate is formed and then spontaneously reacts further to create increasingly more complex products. As such, biomimetic strategies do not rely on the use of sophisticated enzymes, which are usually, but not always, required in a “real” biosynthesis.
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Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family
24.01.2011
Protein Science,
2011,
DOI: 10.1002/pro.594,
656-669
published on 24.01.2011
Protein Science, online article
Protein Science, online article
The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic
domain, CadCpd, is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadCpd (residues 188–512) solved at a resolution of 1.8 A ° via multiple wavelength anomalous dispersion (MAD) using a ReCl6 22 derivative. CadCpd reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a b-sheet in contact with three a-helices and a C-terminal subdomain formed by an eleven-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in
the heavy metal derivative of CadCpd used for MAD phasing a ReCl6 22 ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadCpd forms a homodimer in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.
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Elucidation of the a-Keto-Aldehyde Binding Mechanism: A Lead Structure Motif for Proteasome Inhibition
10.01.2011
Angewandte Chemie,
2011,
50, 2,
542 - 44
published on 10.01.2011
The proteasome's participation in essential biological processes
such as stress response, cell proliferation, apoptosis,
and antigen presentation has been well established.[1] It is,
therefore, not surprising that academia and the pharmaceutical
industry have made efforts to develop a range of small
synthetic inhibitors against this proteolytic molecular
machine (see Scheme SS1 in the Supporting Information for
examples).[2] An overall structural comparison of some wellcharacterized
inhibitors[3] implies that most of these compounds
form a covalent bond with the N-terminal nucleophilic
threonine (Thr1)[4] located at the active sites in the two
inner heptameric b rings of the 20S proteasome, termed beta1,
beta2, and beta5 according to the subunit of their origin.
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New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli
07.01.2011
The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.
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The natural product hybrid of Syringolin A and Glidobactin A synergizes proteasome inhibition potency with subsite selectivity
01.01.2011
Chemical Communications,
2011,
DOI: 10.1039/C0CC02238A, Communication,
385-387
published on 01.01.2011
Chemical Communications, online article
Chemical Communications, online article
The preparation of a Syringolin A/Glidobactin A hybrid (SylA–GlbA) consisting of a SylA macrocycle connected to the GlbA side chain and its potent proteasome targeting of all three proteasomal subsites is reported. The influence of the syrbactin macrocycle moiety on subsite selectivity is demonstrated.
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15.09.2006
To register click here to get to the webpage of the CIPSM-Fest of Biological Chemistry 2011
Program of the Scientific Oktoberfest
Press release of the Scientific Oktoberfest
Pressemitteilung für das Wissenschaftliche Oktoberfest
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