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Biomedical subjects

D F Mierke

Publications and source records attributed to D F Mierke.

At least 55 records · Page 3Linked to original sources
PubMed1996

Secondary structural elements as a basis for antibody recognition in the immunodominant region of human immunodeficiency viruses 1 and 2.

Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV-1 subtype B (1), HIV-2 subtype A (5), and HIV-2 subtype B (7) were synthesized by solid-phase peptide synthesis (Table 1). 1 and 5 were also prepared as their GlcNAc-glycosylated forms at the natural N-glycosylation site NXT (positions 6-8; peptides 4 and 6). Additionally, the proposed beta-turn region of 1 (GPGR; positions 15-18) was altered by introducing D-Ala17 (2) and D-Pro16 (3). All compounds have been studied by two-dimensional NMR techniques. Interproton distances and 3JNH/H alpha coupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE-Distance and angle-bound driven dynamics calculations. The stimulations led to disordered conformations except for a high propensity of a beta II-turn in the region GPXR (positions 15-18) in 1, 2, and 4. In 3 (G-D-ProGR, positions 15-18), a type beta I'-turn was mainly found instead. For peptide 7, the consensus sequence of HIV-2 subtype B, a type beta II-turn was also found although the primary structure (VSGL; positions 15-18) differs grossly from the HIV-1 peptide 1. With the exception of 2, all beta II-turns were able to form a canonically opened beta-turn by a 180 degree rotation of phi(G17). Surprisingly, compounds 5 and 6 that are highly similar to 7 showed no beta II-type turn within MSGL (positions 15-18). They form a type beta VIII-turn across the tetrapeptide SGLV (positions 16-19) together with a non-canonical turn conformation across LMSG (positions 14-17) leading to an S-conformation. The reaction of the peptides with HIV-positive sera from patients infected with different subtypes of HIV-1 and HIV-2 was tested in enzyme-linked immunosorbent assays (ELISA reactions). No HIV-2 sera reacted peptide 1 and no HIV-1 sera showed reactivity to peptide 5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions.

Amino Acid Sequence↗
PubMed1996

Conformational characterization of a peptide mimetic of the third cytoplasmic loop of the G-protein coupled parathyroid hormone/parathyroid hormone related protein receptor.

The third-cytoplasmic loop of the G-protein coupled receptor responsible for the activity of parathyroid hormone and parathyroid hormone-related protein has been structurally characterized in aqueous solution in the presence of sodium dodecylsulfate and dodecylphosphocholine micelles. The high-resolution conformation of the 29-amino acid peptide containing the sequence of the cytoplasmic loop was obtained by CD and nmr. The structure was refined using a two-step distance geometry based method that first includes the removal of all side chains to quickly locate the globular fold of the peptide. After a simulated annealing protocol, the side chains are added in a random fashion. The resulting conformation was further refined with nuclear Overhauser enhancement restrained molecular dynamics using a two-phase simulation cell consisting of carbon tetrachloride and water as a mimetic of the biphasic, hydrophobic/hydrophilic character of the micelles in which the experimental measurements were carried out. The topological orientation of the cycloplasmic loop within the micelle was determined by addition of 5-doxylstearate and monitoring the decrease of nmr signal intensities from the radical-induced relaxation. The conformation and relative orientation of the peptide provided insight into the mechanism by which the cytoplasmic portion of the receptor activates the heterotrimeric, guanine nucleotide-binding regulatory protein, one of the first steps in signal transduction.

Amino Acid Sequence↗
PubMed1994

Molecular structure of the cyanobacterial tumor-promoting microcystins.

The three-dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat-like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.

Amino Acid Sequence↗
PubMed1994

Structure of cobra cardiotoxin CTX I as derived from nuclear magnetic resonance spectroscopy and distance geometry calculations.

The structure of cardiotoxin CTX I from Naja naja atra has been investigated by NMR spectroscopy. Sequence specific resonance assignments have been obtained for all backbone protons as well as for most side-chain protons. Distance geometry calculations were carried out using a metric matrix DG program. A total of 715 NOE constraints, 27 phi angle constraints and a list of the hydrogen bond donors were used for the metric matrix DG calculations and refinement. The average pairwise r.m.s.d. of the resulting structures was 1.01 A for the backbone heavy atoms, and 1.69 A for all heavy atoms. The protein is rich in beta structure and consists of a large triple-stranded, antiparallel beta sheet as well as a short double-stranded, antiparallel beta sheet. Non-regular hydrogen bonding is found between side-chains of the carboxy-terminal end and the rest of the core region. The structure is discussed in terms of evolutionary aspects as well as recent investigations about the biological function and active site.

Amino Acid Sequence↗
PubMed1994

Comparison of the conformation of active and nonactive backbone cyclic analogs of substance P as a tool to elucidate features of the bioactive conformation: NMR and molecular dynamics in DMSO and water.

The conformations of two backbone-cyclized substance P analogs as derived from 1H NMR and molecular dynamics simulations carried out in DMSO and water are described. The method of floating chiralities is used in the simulations to facilitate the diastereotopic assignment of methylene protons. One of the analogs, cyclo-[-(CH2)3-NH-CO-(CH2)4-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is a highly active, selective agonist for the NK-receptor, while the other, cyclo[-(CH2)2-NH-CO-(CH2)2-Gly-Arg-Phe-Phe-N-]-CH2-CO-Leu-Met-NH2, is inactive. Both analogs contain cyclic ring systems of the same size, varying in only the number of amide linkages. From the conformational analysis, the lack of activity can be attributed to the introduction of too much constraint into the ring system. This has an effect on the topological array of the important residues Arg-Phe-Phe. The results presented here are compared with biologically active analogs previously examined. The differences between conformations of active and inactive compounds are used to develop insight into the conformational requirements for biological activity.

Amino Acid Sequence↗
PubMed1994

Coupling constants again: experimental restraints in structure refinement.

Utilization of coupling constants as restraints in computational structure refinement is reviewed. In addition, we address the effect of conformational averaging and examine different approaches to apply the restraints when the experimental observable is obviously a result of averaging. Here, two different computational methods are compared. The simulation of a single structure with time-dependent restraints produces results very similar to those obtained with the calculation of numerous copies of the molecule (an ensemble of structures) and ensemble averaging. The advantages and disadvantages of the two methods are illustrated with simulations of cyclosporin A, for which 117 NOEs and 62 homo- and heteronuclear coupling constants have been measured.

Chemical Phenomena↗
PubMed1994

Coupling constants and hydrogen bonds as experimental restraints in a distance geometry refinement protocol.

A refinement procedure commonly used after distance geometry calculations has been modified to include the use of experimental restraints from coupling constants and hydrogen bonds. Fewer experimental distance constrains (NOEs) are available for peptides as compared to proteins; therefore it is important to incorporate other conformational restraints into refinement methods. The procedure was applied to a cyclic hexapeptide containing two thioamide substitutions, cyclo(-Gly1-Pro2-Phe3 psi [CS-NH]Val4-D-Phe5-Phe6 psi [CS-NH]-). Distance geometry was used to study this peptide, since no potential energy parameters, required in molecular mechanics or dynamics calculations, are available for the thioamide. This is a general problem in the study of peptidomimetics; physiochemical properties of heteroatoms are required within a self-consistent force field. Here, we illustrate the use of metric matrix distance geometry followed by refinement with distance and angle driven dynamics (DADD). We also introduce a new way to handle intramolecular hydrogen bonds by an additional very small and flexible restraint. This method is a viable alternative for the conformational examination of peptides and peptidomimetics. The modifications described here should also find use in the conformational determination of flexible regions of proteins, where the number of NOEs are limited.

Amino Acid Sequence↗
PubMed1993

New evidence for a membrane-bound pathway in hormone receptor binding.

The fully active cholecystokinin analog (Thr,Nle)-CCK-9 was lipo-derivatized by N-terminal grafting of a dimyristoylglycerol moiety to induce tight interdigitation with cell membrane bilayers. While the parent CCK peptide was shown to interact only transiently with small unilamellar phospholipid vesicles, the lipo-CCK peptide, although self-aggregating into vesicles, inserts rapidly and quantitatively into phospholipid bilayers. Fluorescence and, even more so, NMR data are supportive for a chain reversal of the CCK moiety of the lipo derivative with embedment of the C-terminus into hydrophobic compartments of the bilayer. MD simulations allowed for a proposal of the folded form of CCK in bilayers with a helical array parallel to the interface and an amphipathic display of the side chains. In this model, the phenylalanine aromatic ring is heading the peptide molecule and may thus play a decisive role in the lateral penetration of the receptor at the water/lipid interface. In fact, despite the membrane-bound state, its binding affinity for rat pancreatic acini is comparable to that of the CCK peptide when tested after a 3-h equilibration period but 5-6-fold lower at 45 min, suggesting that the association rate is significantly lower than that of the unmodified CCK peptide. This can rationally be attributed to the tight interdigitation of the double-tailed lipo moiety with the membrane bilayer. Moreover, an escape of the lipopeptide into the extracellular aqueous phase is energetically highly unfavored; therefore, the receptor can only be reached by a membrane-bound two-dimensional migration. The observed difference in amplification between binding and amylase secretion may result from inadequate occupation of low-affinity CCK receptors, which leads then to poor couplings to G-proteins. Nevertheless the data confirm that lateral penetration of receptor structures is possible, and thus, preadsorption of peptide (neuro)hormones at the cell membrane bilayer may indeed represent the first step in the receptor recognition process.

Amino Acid Sequence↗
PubMed1993

Synthetic immunogens. The effect of the conformational space on biological and immunological responses to dimeric hormone constructs.

Chimeras of the double chain bis-cystinyl hinge fragment 225-232/225'-232' of the human IgG1 and of peptides related to human little-gastrin were synthesized, whereby the fully bioactive gastrin sequences 2-17 and 5-17 were amide-bond-linked N- and N- or C-terminally, respectively, to the hinge peptide. All the dimeric constructs proved to be efficient immunogens; however, both the configuration of the constructs and the length of the haptenic gastrin molecule were found to drastically affect the specificity of the antibody response and, thus, the type of dominant immune epitope expressed. The different degree of accessibility of the gastrin chains in the dimers is similarly reflected by their binding affinities to gastrin receptors and their bioactivities in vivo. Molecular dynamics simulations of the chimeric compounds clearly revealed that the conformational space of the gastrin peptide chains 2-17 and 5-17 is strongly restricted upon linkage to the hinge peptide. Only in the gastrin-(2-17) construct does sufficient free conformational space seem to be retained, at least for one of the two gastrin chains, in order to allow folding into the bioactive structure. This also agrees with the observation that the dimeric gastrin-(2-17) behaves like a gastrin monomer in terms of receptor binding affinity and biopotency in vivo; but it could additionally explain why an antibody response of gastrin receptor-like specificity could only be induced with this construct. The experimental data may therefore suggest a high degree of parallelism between the mechanism of recognition of the gastrin peptides in the dimeric constructs as hormonal ligands by the gastrin receptors and as haptens by the immune competent cells.

Amino Acid Sequence↗
PubMed1993

Improved molecular dynamics simulations for the determination of peptide structures.

In this article a few methods or modifications proven to be useful in the conformational examination of peptides and related molecules by molecular dynamics are illustrated. The first is the explicit use of organic solvents in the simulations. For many cases such solvents are appropriate since the nmr measurements (or other experimental observations) were carried out in the same solvent. Here, the use of dimethylsulfoxide and chloroform in molecular dynamics is described, with some advantages of the use of these solvents high-lighted. A constant allowing for the scaling of the nonbonded interactions of the force field, an idea previously employed in distance geometry and simulated annealing, has been implemented. The usefulness of this method is that when the nonbonded term is turned to zero, atoms can pass through each other, while the connectivity of the molecule is maintained. It will be shown that such simulations, if a sufficient driving force is present (i.e., nuclear Overhauser effects restraints), can produce the correct stereoconfiguration (i.e., chiral center) as well as configurational isomer (i.e., cis/trans isomers). Lastly, a penalty term for coupling constants directly related to the Karplus curve has been implemented into the potential energy force field. The advantages of this method over the commonly used dihedral angle restraining are discussed. In particular, it is shown that with more than one coupling constant about a dihedral angle a great reduction of the allowed conformational space is obtained.

Algorithms↗
PubMed1992

Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol.

The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and single-step coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to greater than 98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of alpha-helix. Variation of concentration, from 0.5 microM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an alpha-helix extending from Arg19 to Gln34. For the N-terminal half of the molecule no regular structure was observed.

Amino Acid Sequence↗
PubMed1992

The structure of Ro 09-0198 in different environments.

The constitution and configuration of Ro 09-0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09-0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.

Anti-Bacterial Agents↗
PubMed1992

Combined use of homo- and heteronuclear coupling constants as restraints in molecular dynamics simulations.

A penalty function for scalar coupling constants has been applied in molecular dynamics simulations as an experimental constraint. The function is based on the difference between the coupling constant calculated from the dihedral angle and the experimentally measured coupling constant. The method is illustrated on a model cyclic pentapeptide for which 3JHN-H alpha and 3JHN-C beta, both about the phi backbone dihedral angle, have been measured. The function is efficient in producing structures consistent with the scalar couplings, but removed from the conformation observed in solution. This arises from the lack of J restraints for the psi dihedral angle. Simulations with both nuclear Overhauser effect (NOE) and J-coupling restraints illustrates small but significant differences from simulations using only NOEs.

Amino Acid Sequence↗
PubMed1992

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.

Amino Acid Sequence↗
PubMed1992

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. II. Preferred conformations in solution as studied by 1H-NMR spectroscopy.

The 1H-NMR studies were extensively carried out to elucidate preferred conformations of a series of 14-membered cyclic dermorphin analogues containing two phenylalanines at both the third and fourth positions, e.g., Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu], and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu]. The temperature coefficients of the amide proton chemical shifts, vicinal 1H-1H coupling constants for the NH-CH groupings, and nuclear Overhauser effects provided information regarding the preferred conformations of the backbones. The conformational preferences and flexibility of the side chains were also estimated from the vicinal 1H-1H coupling constants around the C-C beta and C beta-C bonds in the articulated side chains. A comparison of the results obtained was made with the results previously obtained for the corresponding enkephalin analogues containing a glycine at the third position. It was found that the replacement of the glycine with the phenylalanine at the third position increases the conformational flexibility of the molecules with an L-, or S-, residue at the fifth position but reduces the flexibility of the molecules with D-, or R-, residue at the same position. The rotating frame nuclear Overhauser experiments gave direct evidence for compact conformations, with the Tyr side chain folding back over the 14-membered ring in Tyr-c[D-Glu-Phe-gPhe-rLeu], which displays relatively high selectivity for the delta-receptor over the mu-receptor. This observation is in agreement with our model proposed for the cyclic enkephalin analogues: folded forms with close aromatic ring placement are required for the activity at the delta-receptor.

Amino Acid Sequence↗
PubMed1990

Conformational studies by circular dichroism, 1H NMR, and computer simulations of bombolitins I and III in aqueous solution containing surfactant micelles.

The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement.

Amino Acid Sequence↗