Biomedical subjects

L Moroder

Publications and source records attributed to L Moroder.

At least 37 records · Page 2Linked to original sources

PubMed2000

Noninvasive tracing of recombinant proteins with "fluorophenylalanine-fingers".

High-level residue-specific replacement of phenylalanine residues in recombinant human annexin V and azurin from Pseudomonas aeruginosa with o-fluorophenylalanine, m-fluorophenylalanine, and p-fluorophenylalanine has been achieved using the selective pressure incorporation method. Incorporation was confirmed analytically and by UV spectroscopy while the secondary and tertiary structures of these protein mutants in solution remained unchanged upon the effected substitutions. Fluorinated phenylalanines alone and when integrated into proteins exhibit two characteristic and prominent shoulders ("fingers") in the UV spectrum in the range of 260-270 nm, which do not overlap with the contributions of tyrosine and tryptophan residues in the protein UV spectra. Thus, the presence of such "fluorophenylalanine fingers" ("FF fingers") opens a new spectral window to identify the labeled target protein among other nonlabeled cellular proteins in preparative work by simple UV spectroscopy. In the coming era of proteomics such a reliable, cheap, and easy reproducible methodology might have a great potential for speeding up the identification and characterization of target molecules in the total protein output from the genomes of a variety of organisms.

Annexin A5↗

PubMed2000

Toward a high-resolution structure of phospholamban: design of soluble transmembrane domain mutants.

Determination of a high-resolution structure of the phospholamban (PLB) transmembrane domain by X-ray crystallography or NMR is handicapped by the hydrophobic nature of the peptide. Interestingly, the crystal structure of the five-stranded parallel coiled-coil oligomerization domain from cartilage oligomeric matrix protein (COMPcc) shows marked similarities to a model proposed for the pentameric transmembrane domain of PLB. Contrary to the putative coiled-coil domain of PLB, COMPcc contains mostly hydrophilic amino acids on the surface, resulting in a soluble molecule. Here, we report the design of soluble PLB transmembrane domain variants by combining the surface residues of COMPcc and the hydrophobic interior of the transmembrane domain of PLB. The soluble PLB variants formed pentameric structures as revealed by analytical ultracentrifugation. After redox shuffling, they showed unspecific disulfide bridge patterns similar to that of the chemically synthesized wild-type PLB transmembrane domain. These results suggest a structural homology between the soluble PLB mutants and the wild-type PLB transmembrane domain. Together with the data reported in the literature, they furthermore indicate that residues Leu37, Ile40, Leu44, and Ile47 of the PLB sequence specify pentamer formation. In contrast, a designed recombinant COMPcc mutant, COMP-ARCC, which was engineered to contain the two PLB cysteines that potentially could form an interchain disulfide bridge, formed a specific disulfide bond pattern. This finding indicates structural differences between the transmembrane domain of PLB and COMPcc. The soluble PLB variants may be used to determine a high-resolution structure of the PLB pentamer by X-ray crystallography.

Amino Acid Sequence↗

PubMed2000

Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor.

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.

Amino Acid Sequence↗

PubMed2000

(4-aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase.

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure-activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N'-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1' enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1'/S2' subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Amino Acid Sequence↗

PubMed2000

Heterotrimeric collagen peptides as fluorogenic collagenase substrates: synthesis, conformational properties, and enzymatic digestion.

The collagenase cleavage site of collagen type I, i.e., the sequence portions 772-784 (P(4)-P(9)') and 772-785 (P(4)-P(10)') of the two alpha1-chains and the sequence portion 772-784 (P(4)-P(9)') of the alpha2-chain, were assembled in an alpha1alpha2alpha1' register by C-terminal cross-linking of these peptides with an artificial cystine knot. The triple-helical conformation of the construct was stabilized by N-terminal extensions with (Gly-Pro-Hyp)(5) repeats. The gaps in the sequence alignment were filled up, and the alpha1-chain was dansylated and the alpha1'-chain was acylated with a tryptophan residue to place in spatial proximity the two chromophores for an efficient fluorescence resonance energy transfer. Although the incorporation of the two N-terminal chromophores leads to partial destabilization of the overall triple-helical fold, the heterotrimer behaved as a collagen-like substrate of the matrix metalloproteinases MMP-1 and MMP-13. Cleavage of the fluorogenic heterotrimer leads to a 6-fold increase in fluorescence intensity, thus making it a useful fluorogenic substrate for interstitial collagenases. With this folded heterotrimeric collagen molecule it was shown that fluorescence resonance energy transfer, as applied so far only for the design of linear fluorogenic enzyme substrates, can also be exploited in conformation dependency.

Animals↗

PubMed2000

Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine.

The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.

Binding Sites↗

PubMed2000

Beta-cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells.

Beta-cyclodextrin is known to form inclusion complexes with hydrophobic drugs. Several tumor cell lines are known to secrete and/or contain membrane-associated cathepsin B which is possibly involved in invasion and metastasis. Based on these information, our recently developed endo-epoxysuccinyl peptide inhibitor MeO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH for cathepsin B was conjugated with beta-cyclodextrin to obtain a site-directed drug carrier system. Furthermore, the conjugate, was shown to form an inclusion complex with the cytotoxic drug methotrexate.

Antimetabolites, Antineoplastic↗

PubMed2000

Synthesis and conformational analysis of apamin analogues with natural and non-natural cystine/selenocystine connectivities.

By replacing two cysteine residues in apamin with selenocysteine, the three possible isomers related to the side-chain connectivities of a bis-cystinyl-peptide were synthesized in regioselective manner exploiting the low redox potential of the diselenide bond. Nuclear magnetic resonance conformational analysis of monoselenocystine analogue apamin with the natural diselenide/disulfide network confirmed the highly isomorphous character of the sulfur replacement with selenium despite its slightly larger atomic radius and increased bond lengths. The comparative conformational analysis of the apamin analogues containing the non-natural side-chain links with wild type apamin clearly revealed retention of the main structural fold and thus the high propensity of these small molecules to adopt the secondary structure elements present in natural apamin. These findings offered interesting hints for a better understanding of the oxidative refolding pathway of the bis-cystinyl peptide that leads exclusively to the correct natural isomer.

Amino Acid Sequence↗

PubMed2000

Synthesis of bivalent inhibitors of eucaryotic proteasomes.

Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X-ray structure of yeast proteasome, homo- and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency. Peptidic bis-aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19-25 monomers was used to link two identical or different tripeptide aldehydes as binding heads. These bis-aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis. Bivalent binding was observed only for the PEG-spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue.

Chymases↗

PubMed2000

Synthesis of beta-(1-azulenyl)-L-alanine as a potential blue-colored fluorescent tryptophan analog and its use in peptide synthesis.

Acetyl-beta-(1-azulenyl)-D,L-alanine has been synthesized in high overall yield by the malonic ester condensation procedure, and the racemate has been enzymatically resolved with acylase I from Aspergillus melleus. The enantiomerically pure L-amino acid is of interest as a blue-colored fluorescent tryptophan analog. The bioactivity data of a heptagastrin analog containing it suggests that the planar aromatic azulene moiety may indeed mimic the tryptophan side chain to some extent, and the spectral properties of the azulene moiety makes beta-(1-azulenyl)-L-alanine of potential value as a UV and fluorescence probe in synthetic peptides, and possibly even in proteins if bioincorporation succeeds with chemically misacylated tRNAs.

Alanine↗

PubMed2000

Photomodulation of conformational states. I. Mono- and bicyclic peptides with (4-amino)phenylazobenzoic acid as backbone constituent.

The thioredoxin reductase active-site fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141], which is known for its high tendency to assume an almost identical conformation as in the intact enzyme, was backbone cyclized with the photoresponsive (4-amino)phenylazobenzoic acid (APB) to produce a monocyclic and disulfide-bridged bicyclic APB-peptide. Light-induced reversible cis/trans isomerization occurs at identical extents in both the linear and the two cyclic forms. Nuclear magnetic resonance conformational analysis clearly revealed that in the bicyclic APB-peptide both as a trans- and cis-azo-isomer the constraints imparted by the bicyclic structure do not allow the molecule to relax into a defined low energy conformation, thus making the molecule a frustrated system that flip-flops between multiple conformational states. Conversely, the monocyclic APB peptide folds into a well-defined lowest energy structure as a trans-azo-isomer, which upon photoisomerization to the cis-azo configuration relaxes into a less restricted conformational space. First femtosecond spectroscopic analysis of the dynamics of the photoreaction confirm a fast first phase on the femtosecond time scale related to the cis/trans isomerization of the azobenzene moiety followed by a slower phase in the picosecond time scale that involves an adjustment of the peptide backbone. Due to the well- defined photoresponsive two-state transition of this monocyclic peptide molecule, it represents a model system well suited for studying the ultrafast dynamics of conformational transitions by time-resolved spectroscopy.

Aminobenzoates↗

PubMed2000

Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent.

It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to an ensemble of possible conformations.

Aminobenzoates↗

PubMed2000

Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases.

BACKGROUND: The general consensus is that interstitial collagens are digested by collagenases and denatured collagen by gelatinases, although processing of fibrillar and acetic-acid-soluble collagen by gelatinase A has also been reported. One of the main difficulties in studying the mechanism of action of these matrix metalloproteinases (MMPs) derives from the physicochemical properties of the natural triple-helical collagen, which makes it difficult to handle. RESULTS: Synthetic heterotrimeric collagenous peptides that contain the collagenase cleavage site of human collagen type I and differ in the thermal stability of the triple-helical fold were used to mimic natural collagen and gelatin, respectively. Results from digestion of these substrates by fibroblast and neutrophil collagenases (MMP-1 and MMP-8), as well as by gelatinase A (MMP-2), confirmed that the two classes of enzymes operate within the context of strong conformational dependency of the substrates. It was also found that gelatinases and collagenases exhibit two distinct proteolytic mechanisms: gelatinase digests the gelatin-like heterotrimer rapidly in individual steps with intermediate releases of partially processed substrate into the medium, whereas collagenases degrade the triple-helical heterotrimer by trapping it until scission through all three alpha chains is achieved. CONCLUSIONS: The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.

Binding Sites↗

PubMed2000

A gated channel into the proteasome core particle.

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme.

Amino Acid Motifs↗

PubMed2000

Urethanyl-3-amidinophenylalanine derivatives as inhibitors of factor Xa. X-ray crystal structure of a trypsin/inhibitor complex and modeling studies.

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the arginine-mimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the X-ray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R)- and (S)-derivatives, combined with modeling experiments, led to an hypothetical non-substrate like binding mode, which was fully confirmed by the remarkably enhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl-(R)-3-amidinophenylalanine-phenethylamide+ ++ a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.

Animals↗

PubMed1999

Atomic mutations at the single tryptophan residue of human recombinant annexin V: effects on structure, stability, and activity.

The single tryptophan residue (Trp187) of human recombinant annexin V, containing 320 residues and 5328 atoms, was replaced with three different isosteric analogues where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine. Such single atom exchanges of H --> F represent atomic mutations that result in slightly increased covalent bond lengths and inverted polarities in the residue side-chain structure. These minimal changes in the local geometry do not affect the secondary and tertiary structures of the mutants, which were identical to those of wild-type protein in the crystal form. But the mutants exhibit significant differences in stability, folding cooperativity, biological activity, and fluorescence properties if compared to the wild-type protein. These rather large global effects, resulting from the minimal local changes, have to be attributed either to the relatively strong changes in polar interactions of the indole ring or to differences in the van der Waals radii or to a combination of both facts. The changes in local geometry that are below resolution of protein X-ray crystallographic studies are probably of secondary importance in comparison to the strong electronegativity introduced by the fluorine atom. Correspondingly, these types of mutations provide an interesting approach to study cooperative functions of integrated residues and modulation of particular physicochemical properties, in the present case of electronegativity, in a uniquely structured and hierarchically organized protein molecule.

Annexin A5↗

PubMed1999

Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor.

Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.

Amino Acid Sequence↗

PubMed1999

Preferred conformation of endomorphin-1 in aqueous and membrane-mimetic environments.

The newly discovered endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are potent opioid peptides with the highest affinity and selectivity for the mu receptor among all known endogenous ligands. To investigate a possible correlation between these biological properties and the conformational preferences of the small peptides, a comparative structural analysis was performed of endomorphin-1 in aqueous buffer and in membrane-mimicking SDS and AOT normal and reverse micelles by the use of CD, FT-IR, fluorescence and(1)H-NMR spectroscopy. It is well established for opioid peptides that, independently of the receptor selectivity, the Tyr1 residue plays the role of the primary pharmacophore and that the orientation of the second aromatic pharmacophore relative to the tyrosine side-chain dictates the mu or delta-receptor selectivity. By varying the environment of endomorphin-1 from water to the amphipathic SDS micelles and even more efficiently to the AOT reverse micelles, the display of the aromatic side-chains changes from an interaction of the Tyr1 and Phe4 residues to a switch of the Trp3 indole group into close contact with the phenolic moiety to prevent this type of interaction and to force an orientation of the Phe4 side-chain into the opposite direction. This conformational switch is accompanied by a stabilization of the cis -Pro2 isomer and the resulting spatial array of the pharmacophoric groups correlate well with the structural model of mu receptor-bound opioid peptides. The results indicate that AOT reverse micelles with a woof 10, where almost exclusively ordered water is secluded in the cavity, constitute with their electrostatic and hydrophobic potential an excellent mimetic of amphipathic surfaces as present on lipid bilayers and on ligand-recognition and ligand-binding sites of proteins.

Circular Dichroism↗