Be inspired -- chemical and functional diversity of new natural product born structures.
Explore the source record for details and available documents.
Combinatorial Chemistry Techniques↗
Biomedical subjects
Ronald Frank
Publications and source records attributed to Ronald Frank.
A new compact disc format of high density array synthesis applied to peptide nucleic acids and in situ MALDI analysis.
A fully automated synthesizer was constructed and designed to perform high speed miniaturized syntheses of compound libraries using the SPOT technique. Utilizing magnetically controlled drop-on-demand ink jet nozzles, an r/phi array format of 2500 spots can be simultaneously dispensed from up to 24 separate reagent valves onto a rotating disc as the solid phase in less than three minutes. In addition, a complete wash station is on board allowing for fully programmable combinatorial syntheses without manual attention. A new carbon black/polypropylene composite solid phase disc was developed and tested for its functionalisation/loading, spot detection, durability and MALDI-TOF target capabilities. The carbon black/polypropylene composite was then successfully employed jointly as the solid phase in the syntheses of short peptide and PNA oligomers and as the target probe holder for MALDI-TOF measurement without transfer of the material. Several protocols for PNA syntheses were also investigated and an optimised PNA methodology for the carbon black/polypropylene composite is reported.
Amino Acid Sequence↗
Defining the antigenic structure of human GM-CSF and its implications for receptor interaction and therapeutic treatments.
Three epitopes of human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF) recognised by neutralising and non-neutralising monoclonal antibodies (mAbs) were characterised using competitive binding assays, dissociation constant measurements with glycosylated and non-glycosylated rhGM-CSF, bioactivity inhibition studies, and synthetic peptide arrays. Based on the first approach, two different binding sites were identified: an area referred to as A, recognised by mAbs M1B8 and CC5B5, and an area referred to as B, recognised by mAbs CC1H7 and CC3C12. These binding sites on hGM-CSF were accurately delineated using cytokine-derived overlapping peptide scans and combinatorial hexapeptide libraries prepared by SPOT synthesis on cellulose membranes. We assigned the identical linear epitope A1P2A3R4 to both non-neutralising mAbs CC1H7 and CC3C12. The conformational epitopes A18E21R23R24F119 and R23E21N17W13 recognised by mAb CC5B5, and P118F119EW13E14 recognised by mAb M1B8, were delineated by dual-positional scanning and subsequent iterative searches with two interrogating positions. Competitive binding assays with mAbs M1B8 and CC5B5 revealed the overlapping nature of the cytokine recognition. However, peptide library screening confirmed their binding to different epitopes of which the essential amino acids were found very closely located on the native protein surface. Inhibition of hGM-CSF biological activity by these mAbs demonstrated that these epitopes are located within or very near the receptor binding site of hGM-CSF. Finally, this work supports the importance of residues from helix A and residues from the C-terminal region of the cytokine, composing a common area that is indispensable for the cytokine's biological activity.
Amino Acid Sequence↗
Multiplexed sorting of libraries on libraries: a novel method for empirical protein design by affinity-driven phage enrichment on synthetic peptide arrays.
Chemically synthesized peptide arrays on planar cellulose carriers are proposed as libraries of ligands suitable for the multiplexed simultaneous capture of peptide-specific acceptor proteins from a large randomly mutagenized library of acceptor proteins presented on bacteriophage M13 particles. This experimental set-up can be exploited to rapidly screen for individual new, distinct binding partners from two complementary libraries (two-dimensional screening). The technical feasibility of this empirical protein design approach was demonstrated with calmodulin as an aceptor protein using an array of mastoparan variants for multiplexed phage affinity enrichment.
Amino Acid Sequence↗
Ectodomains 3 and 4 of human polymeric Immunoglobulin receptor (hpIgR) mediate invasion of Streptococcus pneumoniae into the epithelium.
Streptococcus pneumoniae binds to the ectodomain of the human polymeric Ig receptor (pIgR), also known as secretory component (SC), via a hexapeptide motif in the choline-binding protein SpsA. The SpsA-pIgR interaction mediates adherence and internalization of the human pathogen into epithelial cells. In this study the results of SpsA binding to human, mouse, and chimeric SC strongly supported the human specificity of this unique interaction and suggested that binding sites in the third and fourth Ig-like domain of human SC (D3 and D4, respectively) are involved in SpsA-pIgR complex formation. Binding of SpsA to SC-derived synthetic peptides indicated surface-located potential binding motifs in D3 and D4. Adherence and uptake of pneumococci or SpsA-coated latex beads depended on the SpsA hexapeptide motif as well as SpsA-binding sites in D3 and D4 of human pIgR. The involvement of D3 and D4 in adherence and invasion was demonstrated by the lack of binding of SpsA-coated latex beads to transfected epithelial cells expressing mutated pIgR. Finally, blocking experiments with chimeric human-mouse SC as well as synthetic peptides indicated the participation of D3 and a key role of D4 in pneumococcal invasion.
Amino Acid Motifs↗
Identification and characterization of the Cdc42-binding site of IQGAP1.
IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.
Animals↗
Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.
Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.
Amino Acid Motifs↗
Epitope-targeted proteome analysis: towards a large-scale automated protein-protein-interaction mapping utilizing synthetic peptide arrays.
We describe the development of a process for the genome-wide mapping of interactions between protein domains and peptide ligands entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be "panned" on an array of synthetic peptide ligands. After multiplexed affinity enrichment, peptide-specific phage populations will be automatically eluted, propagated, labelled and identified by hybridisation to a DNA microarray. Peptide arrays are synthesized in situ by SPOT synthesis on a planar substrate. By utilizing a commercially available library of human brain cDNA plus a set of distinct model domains cloned into T7-phage, we could show that a single panning round on an array of known peptide ligands for these model domains synthesized on a cellulose membrane can yield an enrichment of better than a factor of 1,000. This is sufficient to detect peptide-specific enrichment of Cy3(post-panning) against Cy5(pre-panning)-labelled phage DNA inserts on a cDNA microarray. Thus, the proof-of-principle of our approach could be successfully demonstrated and first interaction data are being collected.
Amino Acid Sequence↗
Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae.
The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface-associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal alpha-enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA). The C-terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)-binding site of Eno by investigating the protein-protein interaction. Plasmin(ogen)-binding activity of C-terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno-plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248-256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)-binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing alpha-enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen-binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen-binding motif in the pathogenesis of pneumococcal diseases.
Amino Acid Motifs↗
14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall.
The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists predominantly of Hyp-rich glycoproteins, which also occur in the extracellular matrix of multicellular green algae and higher plants. In addition to the Hyp-rich polypeptides, the insoluble glycoprotein framework of the Chlamydomonas cell wall contains minor amounts of 14-3-3 proteins, as revealed by immunochemical studies and mass spectroscopic analysis of tryptic peptides. Polypeptides immunologically related to the 14-3-3 proteins also were found in the culture medium of Chlamydomonas. The levels of two of these 14-3-3-related polypeptides were decreased in the culture medium of the wall-deficient mutant cw-15. These findings indicate that 14-3-3 proteins are involved in the cross-linking of Hyp-rich glycoproteins in the Chlamydomonas cell wall.
14-3-3 Proteins↗
The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports--principles and applications.
Presented first in 1990 at the 21st European Peptide Symposium in Barcelona, Spain [Frank, R., Güler, S., Krause, S., Lindenmaier, W., 1991. Facile and rapid 'spot synthesis' of large numbers of peptides on membrane sheets. In: Giralt, E., Andreu, D. (Eds.) Peptides 1990, Proc. 21st Eur. Peptide Symp. ESCOM, Leiden, p. 151.], the SPOT-synthesis method opened up countless opportunities to synthesise and subsequently screen large numbers of synthetic peptides as well as other organic compounds arrayed on a planar cellulose support [Tetrahedron 48 (1992) 9217]. Already in 1991, a commercial kit for manual SPOT-synthesis became available through Cambridge Research Biochemicals (CRB, UK), and in 1993, a semi-automated SPOT-synthesiser, the ASP222, was launched by ABIMED Analysen-Technik, Germany. Both made the technique available to many research laboratories, even those not experienced in or equipped for chemistry. Although SPOT-synthesis is not as impressively miniaturised as, e.g. the Affymax photolithographic technique [Science 251 (1991) 767], it fulfils similar demands with the advantage of a reliable and easy experimental procedure, inexpensive equipment needs and a highly flexible array and library formatting. The method permits rapid and highly parallel synthesis of huge numbers of peptides and peptide mixtures (pools) including a large variety of unnatural building blocks, as well as a growing range of other organic compounds. Further advantages are related to the easy adaptability to a wide range of assay and screening methods such as binding, enzymatic and cellular assays, which allow in situ screening of chemical libraries due to the special properties of the membrane supports. Therefore, peptide arrays prepared by the SPOT-technique became quite popular tools for studying numerous aspects of molecular recognition, particularly in the field of molecular immunology.
Combinatorial Chemistry Techniques↗
High-avidity human serum antibodies recognizing linear epitopes of Borna disease virus proteins.
BACKGROUND: The recent observation that Borna disease virus (BDV)-reactive antibodies from psychiatric patients exhibit only low avidity for BDV antigen called into question their diagnostic value and raised the possibility that antigenically related microorganisms or self antigens caused the production of these antibodies. We further characterized the specificity of these antibodies. METHODS: We established a peptide array-based screening test that allows the identification of antibodies directed against linear epitopes of the two major BDV proteins, the nucleoprotein (N) and the phosphoprotein (P). RESULTS: Initial tests employing sera of BDV-infected mice and rats or horses with Borna disease revealed a high specificity and sensitivity of this test. All sera recognized epitopes of N, P, or both. Sera of noninfected rats, mice, and horses showed no signals on either peptide array. Several human sera that recognized BDV antigen by indirect immunofluorescence contained antibodies that recognized various linear epitopes of one or even both BDV proteins. Remarkably, antibodies purified from such human serum by matrix-immobilized peptides showed high-avidity binding to BDV antigens when assayed by IFA or Western blotting. CONCLUSIONS: These data suggest that reactive antibodies found in psychiatric patients indeed indicate infection with BDV or a BDV-like agent. However, the poor affinity maturation of BDV-specific human antibodies remains unexplained.
Animals↗
The Rho-associated protein kinase p160ROCK is required for centrosome positioning.
The p160-Rho-associated coiled-coil-containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis.
Anaphase↗
A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites.
Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat-encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA -proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen-binding motif with the consensus sequence G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X.
Amino Acid Sequence↗
High-density synthetic peptide microarrays: emerging tools for functional genomics and proteomics.
New approaches for manufacturing and application of peptide arrays on planar surfaces are emerging, thereby opening advanced opportunities to probe the expression and function of the proteome. In complementing DNA and protein array analyses, peptide fragment screening directly addresses functional protein interaction sites, leading to a detailed insight into the discovered molecular recognition events, placing them in the context of the whole genome, and even allowing rapid determination of the chemical nature of these interactions. This information can then be transferred into powerful small peptide tools that interfere with these interactions in vivo and help to link targets with phenotypes. With the spreading of new peptide array tools, peptide screening will extend its impact on modern genome-driven molecular biology. This will advance the systematic discovery and validation of new pharmaceutical targets as well as the development of potent molecular diagnostics for medical and ecological monitoring.
Animals↗
Conservation of the amino-terminal epitope of elongation factor Tu in eubacteria and Archaea.
An epitope of elongation factor Tu (EF-Tu), which is found in organisms in both the bacterial and archaeal domains, was recently defined by mAb 900. To localize the conserved epitope within the EF-Tu molecule and to determine its sequence, SPOTScan analysis of synthetic peptides, Western blot analysis of purified EF-Tu domains and site-directed mutagenesis studies were used. Analysis of mAb 900 binding to overlapping 15-mer peptides encompassing the complete sequence of EF-Tu of Escherichia coli was inconclusive, suggesting three distinct regions may be epitopes. Western blot analysis of EF-Tu domains 1-3 of Thermus thermophilus suggested that the epitope was located at the N terminus. This was confirmed by site-directed mutagenesis of EF-Tu domain 1 of Mycoplasma hominis. By C-terminal truncation of the N-terminal 15-mer peptide the epitope was mapped to EF-Tu residues 1-6. Replacement of each of the residues in the epitope peptide demonstrated that only positions 5 and 6 were indispensable for antibody binding. These data provide evidence that the highly conserved epitope recognized by mAb 900 in the bacterial and archaeal domains is located at the very end of the N terminus of the EF-Tu molecule.
Amino Acid Sequence↗
Exploring the mechanisms of vascular smooth muscle tone with highly specific, membrane-permeable inhibitors of cyclic GMP-dependent protein kinase Ialpha.
The structural similarity of cyclic GMP-dependent protein kinase (cGPK) and cyclic AMP-dependent protein kinase (cAPK) has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily due to the lack of potent and selective cGPK inhibitors. We recently reported a novel peptide library screen specifically designed to select for tight-binding peptides that identified selective inhibitors of cGPK [Proc Natl Acad Sci USA, 97 (2000) 14772]. Iterative deconvolution of octameric library arrays on paper identified the sequence LRK(5)H (W45). Binding of W45 to cGPK resulted in selective inhibition of the kinase, with K(i) values of 0.8 microM and 560 microM for cGPK and cAPK, respectively. Cellular internalization of highly charged W45 was accomplished by N-terminal fusion of membrane translocation sequences from either the human immunodeficiency virus tyrosine aminotransferase protein (47-59) DT-2 or from the Drosophila Antennapedia homeodomain (43-58) DT-3, respectively. For both fusion peptides, DT-2 and DT-3, we observed a potentiating effect with respect to the inhibitory potency, with K(i) values 40- to 80-fold lower than W45. Fluorescein-labeled DT-2 and DT-3 demonstrated rapid translocation through the cytosol and nuclei in a time-dependent manner using cultured cells and intact tissue samples (cerebral arteries). The physiological effects of DT-2 and DT-3 as selective cGPK inhibitors in smooth muscle were studied in small intact arteries. Nitric oxide, a cyclic GMP/cGPK activator, elicited a concentration-dependent dilation of isolated rat cerebral arteries, which was markedly inhibited by DT-2 and DT-3. Collectively, these results indicate that DT-2 and DT-3 effectively inhibit nitric oxide-induced vasodilation, further emphasizing the central role for cGPK in the modulation of vascular contractility.
Animals↗