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M Mutter

Publications and source records attributed to M Mutter.

At least 37 records · Page 2Linked to original sources
PubMed1998

Protein design: on the threshold of functional properties.

The ultimate goal in protein de novo design is the creation of novel macromolecules with tailor-made receptor, sensory, and catalytic functions. Despite considerable progress in understanding basic rules of secondary structure formation and protein stability, the well-known protein folding problem is still far from being solved and, in general, only a limited number of designed proteins are folded uniquely. In this article the state-of-the-art in protein design is demonstrated on some selected examples, indicating that the construction of protein-like macromolecules mimicking some essential features of natural proteins seems to be within reach. Thus, protein design and mimicry has become an interdisciplinary challenge with most intriguing perspectives.

Biopolymers↗
PubMed1998

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives.

(S-2-amino-5-(aminooxy)pentanoic acid (L-homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L-homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure.

Aminobutyrates↗
PubMed1998

Mode of action of RG-hydrolase and RG-lyase toward rhamnogalacturonan oligomers. Characterization of degradation products using RG-rhamnohydrolase and RG-galacturonohydrolase.

The mode of action of RG-hydrolase and RG-lyase toward purified linear rhamnogalacturonan (RG) oligomers has been studied. Major tools in the characterization of the degradation products were the exo-acting RG-rhamnohydrolase and RG-galacturonohydrolase. They were used to prepare a series of standards of RG oligomers for HPAEC. 1H NMR spectroscopy confirmed the structure assignment made using HPAEC for a selection of isolated degradation products. Identification of degradation products from purified RG oligomers was then performed by comparing retention times of HPAEC peaks with those of standards. RG-hydrolase was able to cleave RG oligomers which contained five Rha units or more, i.e. DP 9 with a Rha unit at both nonreducing and reducing end. Its preferential cleavage site was at four units from the first nonreducing Rha. RG-lyase was active toward oligomers that contained at least six GalA units, i.e. DP 12 with a GalA at the nonreducing and a Rha at the reducing end. The preferential cleavage site was for the smaller oligomers four residues, and for the largest oligomer six residues from the reducing Rha. From the observed cleavage patterns it can be speculated that in hairy regions, the RG stretches have to be at least 13 residues long for RG-hydrolase and 16 residues long for RG-lyase in order to produce one tetramer.

Chromatography, High Pressure Liquid↗
PubMed1998

Characterization of recombinant rhamnogalacturonan alpha-L-rhamnopyranosyl-(1,4)-alpha-D-galactopyranosyluronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturonan I regions of pectin.

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) alpha-L-rhamnopyranosyl-(1, 4)-alpha-D-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an alpha-Delta-(4,5)-unsaturated D-galactopyranosyluronic acid at the nonreducing end and an L-rhamnopyranose at the reducing end. L-Rhamnopyranose units are substituted at C-4 with beta-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31 degreesC was 28 units mg-1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.

Acetylation↗
PubMed1998

Rhamnogalacturonan alpha-d-galactopyranosyluronohydrolase. An enzyme that specifically removes the terminal nonreducing galacturonosyl residue in rhamnogalacturonan regions of pectin

A new enzyme, rhamnogalacturonan (RG) alpha-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing beta-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 &mgr;m and a maximum reaction rate of 160 units mg-1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50 degreesC. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40 degreesC) and up to 60 degreesC (for 3 h).

Journal Article↗
PubMed1997

Characterization of a selective antagonist of neuropeptide Y at the Y2 receptor. Synthesis and pharmacological evaluation of a Y2 antagonist.

Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 microM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 microM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.

Binding, Competitive↗
PubMed1997

Lysine as helix C-capping residue in a synthetic peptide.

The structure of the synthetic peptide CH3CO(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3Lys-NH2 in trifluoroethanol/water 60/40 (volume ratio) was characterized by two-dimensional nmr spectroscopy. The peptide, closely related to the amphiphilic helix models designed by W. F. De-Grado and co-workers to mimic protein ion channels [(1988) Science, Vol. 240, p. 1177-1181], folds into a regular helix spanning residues 1-20. Evidence for a helix C-terminal capping conformation, involving the terminal lysine residue, was observed from Overhauser effects and checked for consistency by restrained molecular dynamics simulations. The side-chain amino group of Lys22 forms a hydrogen bond with the carbonyl of Leu18, and the distorted helical geometry of the terminal dipeptide allows the inclusion of a water bridge between the backbone NH of the Lys22 residue and the carbonyls of Leu19 and Ser20.

Amino Acid Sequence↗
PubMed1997

Non-native architectures in protein design and mimicry.

Protein design aims to mimic some of the structural and functional properties of native proteins. The complexity of the folding mechanism, i.e. the pathway by which a linear polypeptide chain finds its unique 3D-structure, represents one of the most intriguing hurdles in this rapidly growing field. In order to bypass this well-known protein-folding problem, some years ago we proposed the construction of non-native chain architectures with a high propensity for folding. According to this concept, termed TASP (template-assembled synthetic proteins), topological templates are used as a built-in device for directing covalently attached peptide blocks to a predetermined packing arrangement, resulting in branched chain architectures. Recent progress in the synthetic methodology for assembling peptides now allows us to access the full potential of the TASP concept. In this article, we discuss the state of the art of template-based protein de novo design, with special emphasis on progress in peptide synthesis and template design and show that some fundamental questions in protein assembly, structure and function can be approached by designing protein mimetics of reduced structural and functional complexity.

Drug Design↗
PubMed1997

Isolation and structural characterisation of rhamnogalacturonan oligomers generated by controlled acid hydrolysis of sugar-beet pulp.

Controlled acid hydrolysis was applied to a deesterified beet pulp and the resulting soluble fraction was fractionated on a Biorad AG 1X8 column eluted by ammonium acetate pH 6 from 0.05 to 2 M. Eight retained fractions were obtained, containing almost exclusively GalA and Rha. Three types of oligomers could be identified: homogalacturonans, of which mono-,di- and tri-GalA were isolated as individual components, and two series of rhamnogalacturonan (RG) oligomers. One RG oligomer, isolated after ion-exchange chromatography, was identified as alpha-D-GalAp-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalA p-(1-->2)-L-Rhap. The major peak contained oligomers of dp 6 to more than 20, of which dp 6 to 16 could be isolated on Bio-Gel P-6 + P-4. NMR of the oligomers of dp 6 to 10 showed the following structure: alpha-D-GalAp-(1[-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalA p-(1]n-->2)-L-Rhap. A second, quantitatively minor, series of RG oligomers eluted at higher ionic strength. These oligomers, which could be hydrolysed by RG-hydrolase and RG-lyase, were based on the alternating RG structure. Their non-reducing end was GalA, susceptible to hydrolysis by RG-galacturonohydrolase, and their reducing end might have more than one consecutive GalA.

Acids↗
PubMed1996

Solution structure of regioselectively addressable functionalized templates: an NMR and restrained molecular dynamics investigation.

Three cyclic peptides that are Regioselectively Addressable Functionalized Templates (RAFT) for use in protein de novo design have been investigated using a combination of nmr, restrained molecular dynamics, and CD spectroscopy. These peptides contain up to four selectively addressable sites (orthogonally protected lysine side chains) or have selectively addressable faces. The results show a common stable conformation for templates of this kind based on two type II beta-turns and an associated beta-sheet structure. A preferential orientation for the side chains is also demonstrated. The significance of these findings is discussed in the context of applications of RAFT that rely on their conformational rigidity and ability to present functionalities in a defined spatial arrangement.

Amino Acid Sequence↗
PubMed1996

Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan alpha-L-rhamnopyranosyl-(1-->4)-alpha-D-galactopyranosyluronide lyase.

The recently described rhamnogalacturonase B, which is able to degrade ramified hairy regions of pectin, was found to be a rhamnogalacturonan alpha-L-rhamnopyranosyl-(1-->4)-alpha-D-galactopyranosyluronide lyase. The cleavage site and mechanism differ from that of the previously described rhamnogalacturonase A, which is a hydrolase and can now be termed rhamnogalacturonan alpha-D-galactopyranosyluronide-(1-->2)-alpha-L-rhamnopyranosyl hydrolase.

Aspergillus↗
PubMed1995

A specific template-assembled peptidic agonist for the angiotensin II receptor subtype 2 (AT2) and its effect on inferior olivary neurones.

We synthesized a molecule composed of two angiotensin II 4-8 pentapeptide fragments attached to a carrier molecule (TA), according to the template-assembled synthetic proteins concept. This molecule was investigated for receptor binding on angiotensin type-1 and type-2 receptors (AT1 and AT2) and its biological activity was determined by iontophoretic experiments on neurones of the inferior olive (ION) that express only AT2 receptors. TA binds exclusively to the AT2 receptor and mediates an agonistic angiotensin-II effect on the ION. TA is the first agonist available to study the direct stimulation of AT2 receptors.

Amino Acid Sequence↗
PubMed1995

Templates in protein de novo design.

The de novo design of polypeptide sequences with a three-dimensional structure necessary for many biological functions is limited by the complex folding process, or 'protein folding problem'. This problem can be bypassed through constructing protein-like molecules with a 'built-in' device for intramolecular folding, that is, proteins of non-natural chain architecture (template-assembled synthetic proteins, TASP). Topological templates have become a versatile tool for inducing and stabilizing secondary structures (protein loops, beta-turns, alpha-helices, beta-sheets) and are widely adopted design elements for the construction of protein-like molecules, exhibiting interesting structural and functional properties.

Amino Acid Sequence↗
PubMed1995

Some preliminary results on the action of rhamnogalacturonase on rhamnogalacturonan oligosaccharides from beet pulp.

Sugar-beet pulp was saponified and then hydrolysed with 0.1 M HCl at 80 degrees C for 72 h, and a rhamnogalacturonan fraction was isolated by ion-exchange chromatography on AG 1X8 resin. Four individual oligomers, and a mixture of oligomers with higher degrees of polymerization, were obtained by chromatography on BioGel P-4. They all presented the alpha-D-GalAp-(1[-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalAp -(1]n-->2)-L-Rhap structure (with n > or = 2) The five fractions were submitted to hydrolysis with rhamnogalacturonase. The enzyme was active on oligomers with degrees of polymerization > or = 10, and gave as main products alpha-L-Rhap-(1-->4)-alpha-D-GalAp-(1-->2)-alpha-L-Rhap++ +(1-->4)-D-GalAp and alpha-D-GalAp-(1--2)-alpha-L-Rhap-(1-->4)-alpha-D-GalAp++ +-(1-->2)-alpha-L-Rhap- 1-->4)-D-GalAp.

Carbohydrate Sequence↗
PubMed1994

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein.

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.

Acetylation↗
PubMed1994

Rhamnogalacturonan alpha-L-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin.

Two alpha-L-rhamnohydrolases with different substrate specificities were isolated from a commercial preparation produced by Aspergillus aculeatus. The first rhamnohydrolase was active toward p-nitrophenyl-alpha-L- rhamnopyranoside, naringin, and hesperidin and was termed p-nitrophenyl-alpha-L-rhamnopyranohydrolase (pnp-rhamnohydrolase). From the data collected, the enzyme seemed specific for the alpha-1,2- or alpha-1,6-linkage to beta-D-glucose. The pnp-rhamnohydrolase had a molecular mass of 87 kD (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH optimum of 5.5 to 6, a temperature optimum of 60 degrees C, and a specific activity toward pnp-alpha-L-rhamnopyranoside (pnp-Rha) of 13 units mg-1 protein. The second rhamnohydrolase, on the contrary, was active toward rhamnogalacturonan (RG) fragments, releasing Rha, and was therefore termed RG-rhamnohydrolase. The RG-rhamnohydrolase had a molecular mass of 84 kD, a pH optimum of 4, a temperature optimum of 60 degrees C, and a specific activity toward RG oligomers of 60 units mg-1 protein. The RG-rhamnohydrolase liberated Rha from the nonreducing end of the RG chain and appeared specific for the alpha-1,4-linkage to alpha-D-galacturonic acid. The enzyme was hindered when this terminal Rha residue was substituted at the 4-position by a beta-D-galactose. The results so far obtained did not indicate particular preference of the enzyme for low or high molecular mass RG fragments. From the results it can be concluded that a new enzyme, an RG alpha-L-rhamnopyranohydrolase, has been isolated with high specificity toward RG regions of pectin.

Aspergillus↗