Toward semisynthetic lipoproteins by convergent strategies based on click and ligation chemistry.
Explore the source record for details and available documents.
Chromatography, High Pressure Liquid↗
Biomedical subjects
Luis Moroder
Publications and source records attributed to Luis Moroder.
Isosteric replacement of sulfur with other chalcogens in peptides and proteins.
The review addresses the functional and structural properties of the two series of chalcogen analogues of amino acids in peptides and proteins, the methionine and the serine/cysteine series, and discusses the synthesis of the related selenium/tellurium analogues as well as their use in peptide synthesis and protein expression. Advances in synthetic methodologies and recombinant technologies and their combined applications in native and expressed protein ligation allows the isomorphous character of selenium- and tellurium-containing amino acids to be exploited for production of heavy metal mutants of proteins and thus to facilitate the phasing problem in x-ray crystallography. In addition, selenocysteine has been recognized as an ideal tool for the production of selenoenzymes with new catalytic activities. Moreover, the fully isomorphous character of disulfide replacement with diselenide is well suited to increase the robustness of cystine frameworks in cystine-rich peptides and proteins and for the de novo design of even non-native cystine frameworks by exploiting the highly negative redox potential of selenols.
Chalcogens↗
Studies of protein folding and structure with model peptides.
Explore the source record for details and available documents.
Collagen↗
Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-specific synthetic serine protease inhibitor with weak anticoagulant activity.
Clinical and experimental evidence suggests that the blood coagulation system is involved in the dissemination of malignant tumors. Consequently, anticoagulant agents have been tested as metastasis suppressors in experimental models. Recently, we have found a close correlation between factor Xa (FXa)-specificity of a series of synthetic serine protease inhibitors and their anti-metastatic potential in a murine T-cell lymphoma metastasis model. Interference of such inhibitors with blood-coagulation may represent a major experimental and clinical obstacle. Here, we test anti-metastatic effects of a recently developed, highly specific 3-amidinophenylalanine-type FXa inhibitor, WX-FX4, with weaker anticoagulant activity when compared to well-established FXa inhibitors, such as DX-9065a, as measured by the activated partial thromboplastin time, prothrombin time, prothrombinase complex activity, and coagulation time. Treatment of mice with WX-FX4 (1.5 mg/kg twice daily) led to significant reduction of experimental liver metastasis of a syngeneic T-cell lymphoma in DBA/2 mice (> 90%), and of experimental lung metastasis of a human fibrosarcoma in CD1 nu/nu mice (> 60%). Due to its relatively low anticoagulant activity, daily treatment over 100 days was possible, leading to significant survival benefits without inducing bleeding anomalities. FXa-inhibitors with highly efficient anti-metastatic potential without coagulation-related side effects may represent important new tools as anticancer agents.
Adamantane↗
Incorporation of integrins into artificial planar lipid membranes: characterization by plasmon-enhanced fluorescence spectroscopy.
An optimized peptide-tethered artificial lipid membrane system has been developed. Integrins (cell adhesion receptors) were functionally incorporated into this membrane model and integrin-ligand interactions were analyzed by surface plasmon-enhanced fluorescence spectroscopy (SPFS). The transmembrane receptors alpha(v)beta(3) and alpha(1)beta(1) of the integrin superfamily were incorporated into a lipid-functionalized peptide layer by vesicle spreading. Consecutive layer formations were monitored by surface plasmon spectroscopy (SPS). Orientation and accessibility of the membrane receptor alpha(v)beta(3) was reliably assessed by specific and reproducible binding of selective antibodies. Moreover, full retention of the functional properties of this receptor was verified by specific and reversible binding of natural ligands. Functional integrity of incorporated integrins was maintained over a time period of 72 h. The integrin/extracellular matrix ligand complexes, whose formations are known to depend on the presence of divalent cations, were lost upon addition of ethylenediaminetetraacetate. Therefore, regeneration of the surface for further binding experiments with minimized unspecific ligand association was possible. These results demonstrate that integrins can be functionally incorporated into peptide-tethered artificial membranes. In combination with the SPS/SPFS method, this artificial membrane system provides a reliable experimental platform for investigation of isolated membrane proteins under experimental conditions resembling those of their native environment.
Antibodies↗
Structural properties and photophysical behavior of conformationally constrained hexapeptides functionalized with a new fluorescent analog of tryptophan and a nitroxide radical quencher.
The influence of the conformational properties on the photophysics of two de novo designed hexapeptides was studied by spectroscopic measurements (ir, NMR, steady-state, and time resolved fluorescence) and molecular mechanics calculations. The peptide sequences comprise two nonproteinogenic residues: a beta-(1-azulenyl)-L-alanine (Aal) residue, obtained by formally functionalizing the Ala side chain with the azulene chromophore, and a Calpha-tetrasubstituted alpha-amino acid (TOAC), incorporating a nitroxide group in a cycloalkyl moiety. Aal represents a new fluorescent, quasi-isosteric Trp analog and TOAC a stable radical species, frequently used as a paramagnetic probe in biochemical studies. The peptide chains differ in the sequence position of the two probes and are heavily based on Aib (alpha-aminoisobutyric acid) residues to generate conformationally restricted helical structures, as confirmed by both spectroscopic and computational results. The conformationally controlled, excited state interactions, determining the photophysical relaxation of the Aal*/TOAC pair, are also discussed.
Antioxidants↗
The glycoprotein NOWA and minicollagens are part of a disulfide-linked polymer that forms the cnidarian nematocyst wall.
The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.
Animals↗
A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu(2+) complex.
Since high-intensity synchrotron radiation is available, "extended X-ray absorption fine structure" spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu(2+) complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.
Algorithms↗
Binding mode of TMC-95A analogues to eukaryotic 20S proteasome.
The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.
Binding Sites↗
Crystal structure of NS-134 in complex with bovine cathepsin B: a two-headed epoxysuccinyl inhibitor extends along the entire active-site cleft.
The crystal structure of the inhibitor NS-134 in complex with bovine cathepsin B reveals that functional groups attached to both sides of the epoxysuccinyl reactive group bind to the part of active-site cleft as predicted. The -Leu-Pro-OH side binds to the primed binding sites interacting with the His110 and His111 residues with its C-terminal carboxy group, whereas the -Leu-Gly-Meu (-Leu-Gly-Gly-OMe) part (Meu, methoxycarbonylmethyl) binds along the non-primed binding sites. Comparison with the propeptide structures of cathepsins revealed that the binding of the latter part is least similar to the procathepsin B structure; this result, together with the two-residue shift in positioning of the Leu-Gly-Gly part, suggests that the propeptide structures of the cognate enzymes may not be the best starting point for the design of reverse binding inhibitors.
Animals↗
The G-protein-coupled CCK2 receptor associates with phospholipase Cgamma1.
In ElasCCK2 transgenic mice expressing cholecystokinin (CCK2) receptor in acinar cells, pancreatic phenotypic alterations and preneoplastic lesions are observed. We determined whether activation of phospholipase C gamma1 (PLCgamma1), known to contribute to the tumorigenesis pathophysiology, could take place as a new signaling pathway induced by the CCK2 receptor. Overexpression and activation of the PLCgamma1 in response to gastrin was observed in acinar cells. The possibility that the C-terminal tyrosine 438 of the CCK2 receptor associates with the SH2 domains of PLCgamma1 was examined. A specific interaction was demonstrated using surface plasmon resonance, confirmed in a cellular system and by molecular modeling.
Amino Acid Sequence↗
Crystallographic evidence for isomeric chromophores in 3-fluorotyrosyl-green fluorescent protein.
Explore the source record for details and available documents.
Animals↗
The structure of the Cys-rich terminal domain of Hydra minicollagen, which is involved in disulfide networks of the nematocyst wall.
The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule.
Amino Acid Motifs↗
Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor.
The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.
Amino Acid Sequence↗
A (4R)- or a (4S)-fluoroproline residue in position Xaa of the (Xaa-Yaa-Gly) collagen repeat severely affects triple-helix formation.
The triple-helical fold of collagen requires the presence of a glycine residue at every third position in the peptide sequence and is stabilized by proline and (4R)-4-hydroxyproline residues in positions Xaa and Yaa of the (Xaa-Yaa-Gly) triplets, respectively. Regular down/up puckering of these Xaa/Yaa residues is possibly responsible for the tight packing of the three peptide strands, which have a polyproline-II-like structure, into the supercoiled helix. (4R)-Configured electronegative substituents such as a hydroxy group or a fluorine substituent on the pyrrolidine ring of the residue in the Yaa position favor the up pucker and thus significantly stabilize the triple helix. A similar effect was expected from the corresponding (4S)-isomers in the Xaa positions, but the opposite effect has been observed with (4S)-hydroxyproline, a result that has been speculatively attributed to steric effects. In this study, (4R)- and (4S)-fluoroproline residues were introduced into the Xaa position and potential steric effects were thus avoided. Contrary to expectations, (4S)-fluoroproline prevents triple-helix formation, whereas (4R)-fluoroproline stabilizes the polyPro II conformation, but without supercoiling of the three strands. The latter observation suggests that folding of the single chains into a polyproline II helix is not directly associated with triple helix formation and that fine tuning of van der Waals contacts, electrostatic interactions, and stereoelectronic effects is required for optimal packing into a triple helix.
Amino Acid Sequence↗
Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane.
Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell-adhesion sites that are specifically recognized by cell-surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for alpha1beta1 integrin have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple-helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible alpha2alpha1alpha1' and less probable alpha1alpha2alpha1' chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for alpha1beta1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod-shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix.
Basement Membrane↗
Micellar environments induce structuring of the N-terminal tail of the prion protein.
In the physiological form, the prion protein is a glycoprotein tethered to the cell surface via a C-terminal glycosylphosphatidylinositol anchor, consisting of a largely alpha-helical globular C-terminal domain and an unstructured N-terminal portion. This unstructured part of the protein contains four successive octapeptide repeats, which were shown to bind up to four Cu(2+) ions in a cooperative manner. To mimic the location of the protein on the cell membrane and to analyze possible structuring effects of the lipid/water interface, the conformational preferences of a single octapeptide repeat and its tetrameric form, as well of the fragment 92-113, proposed as an additional copper binding site, were comparatively analyzed in aqueous and dodecylphosphocholine micellar solution as a membrane mimetic. While for the downstream fragment 92-113 no conformational effects were detectable in the presence of DPC micelles by CD and NMR, both the single octapeptide repeat and, in an even more pronounced manner, its tetrameric form are restricted into well-defined conformations. Because of the repetitive character of the rigid structural subdomain in the tetrarepeat molecule, the spatial arrangement of these identical motifs could not be resolved by NMR analysis. However, the polyvalent nature of the repetitive subunits leads to a remarkably enhanced interaction with the micelles, which is not detectably affected by copper complexation. These results strongly suggest interactions of the cellular form of PrP (PrP(c)) N-terminal tail with the cell membrane surface at least in the octapeptide repeat region with preorganization of these sequence portions for copper complexation. There are sufficient experimental facts known that support a physiological role of copper complexation by the octapeptide repeat region of PrP(c) such as a copper-buffering role of the PrP(c) protein on the extracellular surface.
Amino Acid Sequence↗
TMC-95A analogues with endocyclic biphenyl ether group as proteasome inhibitors.
TMC-95A, a cyclic tripeptide metabolite of Apiospora montagnei, is a potent competitive inhibitor of proteasome. Based on the X-ray structure of its complex with yeast proteasome, the synthetically challenging structure of this natural product was simplified in a first generation of analogues by replacing the highly oxidized side-chain biaryl system with a phenyl-oxindole group. In the present study, the TMC-95 biaryl group was substituted with a biphenyl ether with retainment of significant proteasome inhibition. Because of the facile synthetic access of tripeptides containing in i, i+2 positions residues of the isodityrosine type, this new generation of TMC-95 analogues may represent promising lead structures for further optimization of affinity and selectivity of proteasome inhibitors.
Biphenyl Compounds↗