Container molecules with portals: Reversibly switchable cycloalkane complexation.
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
Bernhard Jaun
Publications and source records attributed to Bernhard Jaun.
Simulation of beta-depsipeptides: the effect of missing hydrogen-bond donors on their folding equilibria.
beta-Depsipeptides are beta-peptides in which one or more peptide linkages are replaced by ester linkages, resulting in a loss of a hydrogen-bond donor (N--H) and weakening of the corresponding carbonyl hydrogen-bond acceptor moiety. The effects of three of such peptide by ester substitutions in a hepta-beta-peptide upon its (un)folding equilibrium in methanol solution are investigated using molecular dynamics simulations and compared to experimental data from NMR spectroscopy. The simulated conformational ensembles largely reproduce the experimentally measured NOE and 3J-coupling constant data for the three different hepta-beta-peptides, and confirm the relative stabilities of the 3(14)-helical conformation, which is most weakened by substitution of the 4th peptide linkage and least by substitution of the 6th peptide linkage. The simulations are complementary to the experimental data by providing detailed insight into the conformational distributions that are compatible with the experimentally measured average values of observables.
Computational Biology↗
Simulation of an all-beta 3-icosapeptide containing the 20 proteinogenic side chains: effect of temperature, pH, counterions, solvent, and force field on helix stability.
Simulations of various beta-peptides have in the last years clarified several issues concerning peptide folding equilibria and interpretation of experimental data, especially from NMR and CD spectroscopy. These simulations involved different temperatures, pH-values, ionic strengths, solvents, and force-field parameters, but a variation of these factors for one beta-peptide has not yet been done. To investigate the influence of varying these factors, we analyze the helix stability of an all-beta3-icosapeptide bearing all 20 proteinogenic amino acid side chains, which is experimentally observed to fold into a 3(14)-helix in methanol but not in water. Structural aspects, such as hydrogen-bonded rings and salt bridges, are discussed and a comparison with NMR primary (NOE distance bounds and 3J-values) and secondary (NMR derived model structures) data is made. We further investigate the reasons for the 3(14)-helix stability/instability in methanol/water. Of all factors studied, the presence of counterions seems to be the one inducing most significant effects in the simulations.
Computer Simulation↗
A nickel-alkyl bond in an inactivated state of the enzyme catalyzing methane formation.
Explore the source record for details and available documents.
Alkanesulfonic Acids↗
Spin density and coenzyme M coordination geometry of the ox1 form of methyl-coenzyme M reductase: a pulse EPR study.
Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR <--> Ni(II)-*SR.
Electron Spin Resonance Spectroscopy↗
On the influence of charged side chains on the folding-unfolding equilibrium of beta-peptides: a molecular dynamics simulation study.
The influence of charged side chains on the folding-unfolding equilibrium of beta-peptides was investigated by means of molecular dynamics simulations. Four different peptides containing only negatively charged side chains, positively charged side chains, both types of charged side chains (with the ability to form stabilizing salt bridges) or no charged side chains were studied under various conditions (different simulation temperatures, starting structures and solvent environment). The NMR solution structure in methanol of one of the peptides (A) has already been published; the synthesis and NMR analysis of another peptide (B) is described here. The other peptides (C and D) studied herein have hitherto not been synthesized. All four peptides A-D are expected to adopt a left-handed 3(14)-helix in solution as well as in the simulations. The resulting ensembles of structures were analyzed in terms of conformational space sampled by the peptides, folding behavior, structural properties such as hydrogen bonding, side chain-side chain and side chain-backbone interactions and in terms of the level of agreement with the NMR data available for two of the peptides. It was found that the presence of charged side chains significantly slows down the folding process in methanol solution due to the stabilization of intermediate conformers with side chain-backbone interactions. In water, where the solvent competes with the solute-solute polar interactions, the folding process to the 3(14)-helix is faster in the simulations.
Hydrogen Bonding↗
Interpreting NMR data for beta-peptides using molecular dynamics simulations.
NMR is one of the most used techniques to resolve structure of proteins and peptides in solution. However, inconsistencies may occur due to the fact that a polypeptide may adopt more than one conformation. Since the NOE distance bounds and (3)J-values used in such structure determination represent a nonlinear average over the total ensemble of conformers, imposition of NOE or (3)J-value restraints to obtain one unique conformation is not an appropriate procedure in such cases. Here, we show that unrestrained MD simulation of a solute in solution using a high-quality force field yields a conformational ensemble that is largely compatible with the experimental NMR data on the solute. Four 100 ns MD simulations of two forms of a nine-residue beta-peptide in methanol at two temperatures produced conformational ensembles that were used to interpret the NMR data on this molecule and resolve inconsistencies between the experimental NOEs. The protected and unprotected forms of the beta-peptide adopt predominantly a 12/10-helix in agreement with the qualitative interpretation of the NMR data. However, a particular NOE was not compatible with this helix indicating the presence of other conformations. The simulations showed that 3(14)()-helical structures were present in the ensemble of the unprotected form and that their presence correlates with the fulfillment of the particular NOE. Additionally, all inter-hydrogen distances were calculated to compare NOEs predicted by the simulations to the ones observed experimentally. The MD conformational ensembles allowed for a detailed and consistent interpretation of the experimental data and showed the small but specific conformational differences between the protected and unprotected forms of the peptide.
Computer Simulation↗
Direct interaction of coenzyme M with the active-site Fe-S cluster of heterodisulfide reductase.
Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S=1/2 [4Fe-4S]3) cluster, the EPR spectrum of which is broadened in the presence of CoM-33SH [Duin, E.C., Madadi-Kahkesh, S., Hedderich, R., Clay, M.D. and Johnson, M.K. (2002) Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. FEBS Lett. 512, 263-268; Duin, E.C., Bauer, C., Jaun, B. and Hedderich, R. (2003) Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme. FEBS Lett. 538, 81-84]. These results provide indirect evidence that the disulfide binds to the iron-sulfur cluster during reduction. We report here direct structural evidence for this interaction from Se X-ray absorption spectroscopic investigation of HDR treated with the selenium analog of coenzyme M (CoM-SeH). Se K edge extended X-ray absorption fine structure confirms a direct interaction of the Se in CoM-SeH-treated HDR with an Fe atom of the Fe-S cluster at an Fe-Se distance of 2.4A.
Binding Sites↗
Mannich-type C-nucleosidations in the 5,8-diaza-7,9-dicarba-purine family.
[structure: see text] C-Nucleosidation with cyclic iminium salts occurring under mild reaction conditions and affording C-nucleosides that are isosteric with N-nucleosides of natural purines is shown to be a consistent property of the entire family of 2,6-(oxo or amino)-disubstituted 5,8-diaza-7,9-dicarba-purines.
Magnetic Resonance Spectroscopy↗
Peralkynylated buta-1,2,3-trienes: exceptionally low rotational barriers of cumulenic C=C bonds in the range of those of peptide C--N bonds.
A variety of 1,1,4,4-tetraal kynylbutatrienes and 1,4-dialkynylbutatrienes was synthezized by dimerization of the corresponding gem-dibromoolefins. Both (1)H and (13)C NMR spectroscopy indicated that the di- and tetraalkynylated butatrienes are formed as a mixture of cis and trans isomers. Variable temperature NMR studies evidenced a facile cis-trans isomerization, thus preventing the separation of these isomers by gravity or high-performance liquid chromatography (HPLC). For 1,1,4,4-tetraalkynylbutatrienes, the activation barrier deltaG( not equal ) was measured by magnetization transfer to be around 20 kcal mol(-1), in the range of the barrier for internal rotation about a peptide bond. Unlike the tetraalkynylated [3]cumulenes, 1,4-dialkynylbutatrienes are more difficult to isomerize and could, in one case, be obtained isomerically pure. Based on experimental data, the rotational barrier DeltaG( not equal ) for 1,4-dialkynylbutatrienes is estimated to be around 25 kcal mol(-1). The hypothesis of a stabilizing effect of the four alkynyl substituents on the proposed but-2-yne-1,4-diyl singlet diradical transition state of this cis-trans isomerization is further supported by a computational study.
Alkenes↗
Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues.
Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F(430) as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K(M)=20 mM; apparent V(max)=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K(M)=5 mM; apparent V(max)=30 U/mg). Propyl-coenzyme M (apparent K(i)=2 mM) and allyl-coenzyme M (apparent K(i)=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I](0.5 V)=2 micro M), cyano-coenzyme M ([I](0.5 V)=0.2 mM), 3-bromopropionate ([I](0.5 V)=3 mM), seleno-coenzyme M ([I](0.5 V)=6 mM) and trifluoromethyl-coenzyme M ([I](0.5 V)=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D(2)O. The signal was abolished upon exposure of the enzyme to O(2). 3-Bromopropanesulfonate ([I](0.5 V)=0.1 micro M), 3-iodopropanesulfonate ([I](0.5 V)=1 micro M), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O(2). 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.
Alkanesulfonic Acids↗
Spectroscopic investigation of the nickel-containing porphinoid cofactor F(430). Comparison of the free cofactor in the (+)1, (+)2 and (+)3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent, red and ox forms.
Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F(430), a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F(430) in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F(430), variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F(430)M) in the (+)1, (+)2 and (+)3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle pi orbitals that are unique to Ni(I) forms of F(430). Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d-d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F(430) macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.
Archaea↗
Design, synthesis and structural investigations of a beta-peptide forming a 314-helix stabilized by electrostatic interactions.
Two different strategies have been employed for the synthesis of Fmoc-protected beta(3)-homoarginine; the Arndt-Eistert homologation of alpha-arginine and the guanidinylation of beta(3)-homoornithine. Solid-phase beta-peptide synthesis was used for the preparation of beta-heptapeptide 1, which was designed to form a helix stabilized by electrostatic interactions through positively (beta(3)hArg) and negatively charged (beta(3)hGlu) amino acid residues. CD measurements and corresponding NMR investigations in MeOH and aqueous solutions do indeed show that the beta-peptidic 3(14)-helix can be stabilized by salt-bridge formation.
Arginine↗
Molecular dynamics simulations of small peptides: can one derive conformational preferences from ROESY spectra?
Folding properties of beta-peptides were investigated by means of NMR experiments and MD simulations of beta-dipeptides, which serve as small test systems to study the influence of stereocenters and side chains on hydrogen-bond and consequently on secondary-structure formation. Two stereoisomers, SR and SS, of a Val-Phe dipeptide, and of the corresponding Ala-Ala dipeptide, and a Gly-Gly dipeptide were simulated in methanol for 40 ns. In agreement with experiment, the isomers of the Val-Phe dipeptide adopt quite different conformers at 298 K, the differences being reduced at 340 K. Interestingly, the SR isomer shows enhanced hydrogen bonding at the higher temperature. The adopted conformations are primarily determined by the R or S side chain substitution, and less by the type of side chain. Back-calculation of (1)H ROESY spectra and (3)J coupling constants from the MD simulations and comparison with the experimental data for the Val-Phe dipeptides shows good agreement between simulation and experiment, and reveals possible problems and pitfalls, when deriving structural properties of a small and extremely flexible molecule from NMR data only. Inclusion of all aspects of internal dynamics is essential to the correct prediction of the NMR spectra of these small molecules. Cross comparison of calculated with experimental spectra for both isomers shows that only a few out of many ROESY peaks reflect the sizeable conformational differences between the isomers at 298 K.
Dipeptides↗
Direct determination of the number of electrons needed to reduce coenzyme F430 pentamethyl ester to the Ni(I) species exhibiting the electron paramagnetic resonance and ultraviolet-visible spectra characteristic for the MCR(red1) state of methyl-coenzyme M reductase.
The UV-visible and electron paramagnetic resonance (EPR) spectra of MCR(red1), the catalytically active state of methyl-coenzyme M reductase, are almost identical to those observed when free coenzyme F430 or its pentamethyl ester (F430M) are reduced to the Ni(I) valence state. Investigations and proposals concerning the catalytic mechanism of MCR were therefore based on MCR(red1) containing Ni(I)F430 until, in a recent report, Tang et al. (J. Am. Chem. Soc. 2002, 124, 13242) interpreted their resonance Raman data and titration experiments as indicating that, in MCR(red1), coenzyme F430 is not only reduced at the nickel center but at one of the C=N double bonds of the hydrocorphinoid macrocycle as well. To resolve this contradiction, we have investigated the stoichiometry of the reduction of coenzyme F430 pentamethyl ester (F430M) by three independent methods. Spectroelectrochemistry showed clean reduction to a single product that exhibits the UV-vis spectrum typical for MCR(red1). In three bulk electrolysis experiments, 0.96 +/- 0.1 F/mol was required to generate the reduced species. Reduction with decamethylcobaltocene in tetrahydrofuran (THF) consumed 1 mol of (Cp)(2)Co/mol of F430M, and the stoichiometry of the reoxidation of the reduced form with the two-electron oxidant methylene blue was 0.46 +/- 0.05 mol of methylene blue/mol of reduced F430M. These experiments demonstrate that the reduction of coenzyme F430M to the species having almost identical UV-vis and EPR spectra as MCR(red1) is a one-electron process and therefore inconsistent with a reduction of the macrocycle chromophore.
Electrochemistry↗
C-Nucleosidations with 2,6-diamino-5,8-diaza-7,9-dicarba-purine.
[structure: see text] Endocyclic iminium ions derived from l-4-amino-threose derivatives smoothly react with 2,6-diamino-5,8-diaza-7,9-dicarba-purine to give corresponding C(9)-nucleosides in high yields.
2-Aminopurine↗
Coenzyme B induced coordination of coenzyme M via its thiol group to Ni(I) of F430 in active methyl-coenzyme M reductase.
Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.
Electron Spin Resonance Spectroscopy↗
The fourth helical secondary structure of beta-peptides: the (P)-28-helix of a beta-hexapeptide consisting of (2R,3S)-3-amino-2-hydroxy acid residues.
Explore the source record for details and available documents.
Amino Acids↗