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T de Beer

Publications and source records attributed to T de Beer.

17 recordsLinked to original sources
PubMed2002

Laser-based non-fluorescence detection techniques for liquid separation systems.

Over the last two decades, the possibility to use lasers for detection purposes in column liquid chromatography (LC) and capillary electrophoresis (CE) received much attention in the analytical chemistry literature. Most attention has been devoted to laser-induced fluorescence. The present review covers developments on non-fluorescence techniques for LC and CE. The techniques considered are thermal lens spectrometry, photoacoustic detection, refractive index detection including refractive index backscattering, Raman spectroscopy and degenerate four-wave mixing (a special mode of transientholographic spectroscopy). The paper starts with an outline of the characteristics of lasers; it ends with an overall evaluation and a discussion of the perspectives of the techniques dealt with.

Lasers↗
PubMed2001

Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes.

Specific recognition of phosphoinositides is crucial for protein sorting and membrane trafficking. Protein transport to the yeast vacuole depends on the Vam7 t-SNARE and its phox homology (PX) domain. Here, we show that the PX domain of Vam7 targets to vacuoles in vivo in a manner dependent on phosphatidylinositol 3-phosphate generation. A novel phosphatidylinositol-3-phosphate-binding motif and an exposed loop that interacts with the lipid bilayer are identified by nuclear magnetic resonance spectroscopy. Conservation of key structural and binding site residues across the diverse PX family indicates a shared fold and phosphoinositide recognition function.

Amino Acid Sequence↗
PubMed2000

Solution structure of Eps15's third EH domain reveals coincident Phe-Trp and Asn-Pro-Phe binding sites.

Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Molecular mechanism of NPF recognition by EH domains.

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH-NPF interaction, the solution structures of two EH-NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I beta-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the beta-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.

Amino Acid Motifs↗
PubMed1999

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.

Bacteriophage lambda↗
PubMed1999

Phosphatidylinositol 3-phosphate recognition by the FYVE domain.

Recognition of phosphatidylinositol 3-phosphate (Ptdlns(3)P) is crucial for a broad range of cellular signaling and membrane trafficking events regulated by phosphoinositide (PI) 3-kinases. PtdIns(3)P binding by the FYVE domain of human early endosome autoantigen 1 (EEA1), a protein implicated in endosome fusion, involves two beta hairpins and an alpha helix. Specific amino acids, including those of the FYVE domain's conserved RRHHCRQCGNIF motif, contact soluble and micelle-embedded lipid and provide specificity for Ptdlns(3)P over Ptdlns(5)P and Ptdlns, as shown by heteronuclear magnetic resonance spectroscopy. Although the FYVE domain relies on a zinc-binding motif reminiscent of RING fingers, it is distinguished by ovel structural features and its ptdlns(3)P-binding site.

Amino Acid Sequence↗
PubMed1998

Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain.

Eps15 homology (EH) domains are eukaryotic signaling modules that recognize proteins containing Asn-Pro-Phe (NPF) sequences. The structure of the central EH domain of Eps15 has been solved by heteronuclear magnetic resonance spectroscopy. The fold consists of a pair of EF hand motifs, the second of which binds tightly to calcium. The NPF peptide is bound in a hydrophobic pocket between two alpha helices, and binding is mediated by a critical aromatic interaction as revealed by structure-based mutagenesis. The fold is predicted to be highly conserved among 30 identified EH domains and provides a structural basis for defining EH-mediated events in protein trafficking and growth factor signaling.

Amino Acid Sequence↗
PubMed1998

Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the alpha-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free alpha-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free alpha-subunit. For accurate modeling of alpha hCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free alpha-subunit (as-pd alpha) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing alpha hCG. From the values of 13C relaxation parameters T2 and T1 rho of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4' residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4' branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide.

Asparagine↗
PubMed1996

Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms.

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH > 5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.

Amino Acid Sequence↗
PubMed1996

Polydispersity in sulfation profile of oligosaccharide alditols isolated from the protein-linkage region and the repeating disaccharide region of chondroitin 4-sulfate of bovine nasal septal cartilage.

Proteoglycans of bovine nasal septal cartilage bear predominantly chondroitin 4-sulfate. After exhaustive chondroitinase ABC digestion of a chondromucoprotein preparation rich in proteoglycans and subsequent reductive beta-elimination, five hexasaccharide alditols were isolated from the glycosaminoglycan-protein linkage region. They were analyzed by enzymatic digestion in conjunction with HPLC and by one-dimensional and two-dimensional 1H-NMR spectroscopy. They share the conventional core saccharide structure delta 4.5HexA alpha 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol (where delta 4.5HexA is 4,5-unsaturated hexuronic acid), but have different sulfation profiles. One compound (I) does not contain sulfate. Two of the three monosulfated compounds (II and III) have an O-sulfate group at either C6 or at C4 of the GalNAc residue. The other monosulfated compound (IV) is hitherto unreported and has a O-sulfate at C4 of the Gal residue preceding the GlcA residue, whereas the GalNAc is not sulfated. The disulfated compound (V) has sulfate groups at C4 of both the Gal residue preceding GlcA and the GalNAc residue. The molar ratio of compounds I-V is 38.3:5.9:43.0:1.6:11.2. The structural heterogeneity of these hexasaccharide alditols reflects the polydispersity in the linkage region of the chondroitin sulfate chains. In addition, two trisaccharide and two tetrasaccharide alditols derived from the repeating disaccharide region of the chondroitin sulfate chains were also isolated. Their structures were unambiguously determined by enzymatic analysis and by 1H-NMR spectroscopy as delta 4.5HexA alpha 1-3GalNAc(4-O- or 6-O-sulfate)beta 1-4GlcA-ol and delta 4.5HexA alpha 1-3GalNAc(4-O- or 6-O-sulfate) beta 1-4GlcA beta 1-3GalNAc(4-O-sulfate)-ol, respectively.

Animals↗
PubMed1995

Structure determination of the octa- and decasaccharide sequences isolated from the carbohydrate-protein linkage region of porcine intestinal heparin.

Previously we isolated a tetrasaccharide-serine and a hexasaccharide-serine from the carbohydrate-protein linkage region of porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II (Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J.F.G. (1992) J. Biol. Chem. 267, 1528-1533). In this study four longer carbohydrate sequences (I-IV) attached to Ser or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2% of the total linkage region, were isolated from the same heparin preparation after digestion with heparinase only. IV was successfully isolated only after subsequent digestion with glycuronate-2-sulfatase. Their structures were determined by chemical and enzymatic analyses and 1H NMR spectroscopy and found to be the following octa- and decasaccharide sequences attached to Ser in a molar ratio of 1.1:2.3:1.0:1.3: delta HexA(2S)alpha 1-4GlcN(NS,6S)alpha 1-4GlcA beta 1-4GlcNAc alpha 1-4- GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (I), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1- 3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (II), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1- 4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcNAc-alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (III), delta HexA alpha 1-4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc(6S)alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (IV) (delta HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated hexuronic acid, D-glucuronic acid, L-iduronic acid, and D-glucosamine, whereas 2S, 6S, and NS stand for 2-sulfate, 6-sulfate, and N-sulfate, respectively). I and II contained 1 mol of Gly in addition to Ser. The four structures indicate that sulfation in heparin chains takes place on the monosaccharide residues located in closer vicinity to the core protein than found for heparan sulfate chains and that there exist at least several heparin subclass chains with different linkage region structures. The significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of heparin.

Amino Acids↗
PubMed1995

The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is alpha-mannopyranose.

Recently, the novel C-glycosidic linkage of a hexopyranosyl residue to the indole ring of tryptophan residue 7 of human RNase U(s) was reported [Hofsteenge, J., Müller, D. R., de Beer, T., Löffler A., Richter, W. J., & Vliegenthart, J. F. G. (1994) Biochemistry 33, 13524-13530]. Identification of this monosaccharide is a prerequisite for studies of its biosynthesis and its biological relevance. Using vicinal proton-proton coupling constants and rotating-frame nuclear Overhauser enhancements, ewe demonstrate that the C-linked substituent is alpha-mannopyranose. Furthermore, the nuclear magnetic resonance (NMR) data indicate that the mannopyranose moiety in a glycopeptide derived from RNase U(s) adopts several conformations on the NMR time scale.

Amino Acid Sequence↗
PubMed1994

New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.

We report a new type of linkage between a carbohydrate and a protein, involving the rarely modified side chain of a tryptophan residue. An aldohexopyranosyl residue was found to be linked via a C-C bond to the indole ring of the tryptophan residue at position 7 of human RNase Us. Mass spectrometric analysis of peptides containing this residue showed a molecular mass 162 Da higher than that expected for tryptophan. The fragmentation pattern of the modified amino acid side chain was reminiscent of that of aromatic C-glycosides, suggesting a direct attachment of a hexose residue to a C-position of the tryptophan indole moiety. 1H and 13C NMR spectroscopic data confirmed this inference and unequivocally demonstrated the substituent to be an aldohexopyranosyl residue, C-glycosidically linked to the C2 atom of the indole. This mode of attachment differs from the ones known so far, in which carbohydrates are linked to an amino acid side chain by N- or O-glycosidic bonds.

Amino Acid Sequence↗
PubMed1994

Rapid and simple approach for the NMR resonance assignment of the carbohydrate chains of an intact glycoprotein. Application of gradient-enhanced natural abundance 1H-13C HSQC and HSQC-TOCSY to the alpha-subunit of human chorionic gonadotropin.

The structure assessment of an intact glycoprotein in solution requires an extensive assignment of the carbohydrate NMR resonances. However, assignment of homonuclear spectra is very complicated because of the severe overlap of protein and carbohydrate signals. Application of pulsed field gradients allowed high quality natural abundance 1H-13C HSQC and HSQC-TOCSY spectra to be recorded of the alpha-subunit of human chorionic gonadotropin. Most carbohydrate 1H-13C correlations appear in a distinct region between the aromatic region and the protein C alpha-H alpha region. The enormous reduction in overlap led to fast and unambiguous assignment of the anomeric 1H-13C correlations. Subsequently, correlations of the monosaccharide skeleton atoms were readily assigned in the HSQC-TOCSY spectrum.

Carbohydrate Sequence↗