Comparative Dynamics of Methionine Side-Chain in FMOC-Methionine and in Amyloid Fibrils.
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PROVIDER: S-EPMC5612497 | BioStudies |
REPOSITORIES: biostudies
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Project description:Reactive oxygen species (ROS) cause damage to DNA and proteins. Here, we report that the RecA recombinase is itself oxidized by ROS. Genetic and biochemical analyses revealed that oxidation of RecA altered its DNA repair and DNA recombination activities. Mass spectrometry analysis showed that exposure to ROS converted four out of nine Met residues of RecA to methionine sulfoxide. Mimicking oxidation of Met35 by changing it for Gln caused complete loss of function, whereas mimicking oxidation of Met164 resulted in constitutive SOS activation and loss of recombination activity. Yet, all ROS-induced alterations of RecA activity were suppressed by methionine sulfoxide reductases MsrA and MsrB. These findings indicate that under oxidative stress MsrA/B is needed for RecA homeostasis control. The implication is that, besides damaging DNA structure directly, ROS prevent repair of DNA damage by hampering RecA activity.
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Project description:A synthetic strategy for the preparation of two orthogonally protected methyl esters of the non-proteinogenic amino acid 2,3-l-diaminopropanoic acid (l-Dap) was developed. In these structures, the base-labile protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was paired to the <i>p</i>-toluensulfonyl (tosyl, Ts) or acid-labile <i>tert</i>-butyloxycarbonyl (Boc) moieties. The synthetic approach to protected l-Dap methyl esters uses appropriately masked 2,3-diaminopropanols, which are obtained via reductive amination of an aldehyde prepared from the commercial amino acid <i>N</i><sup>?</sup>-Fmoc-<i>O</i>-<i>tert</i>-butyl-d-serine, used as the starting material. Reductive amination is carried out with primary amines and sulfonamides, and the process is assisted by the Lewis acid Ti(O<sup>i</sup>Pr)<sub>4</sub>. The required carboxyl group is installed by oxidizing the alcoholic function of 2,3-diaminopropanols bearing the tosyl or benzyl protecting group on the 3-NH<sub>2</sub> site. The procedure can easily be applied using the crude product obtained after each step, minimizing the need for chromatographic purifications. Chirality of the carbon atom of the starting d-serine template is preserved throughout all synthetic steps.
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Project description:Aromatic residues are important markers of dynamical changes in proteins' hydrophobic cores. In this work we investigated the dynamics of the F19 side-chain in the core of amyloid fibrils across a wide temperature range of 300 to 140 K. We utilized solid-state 2H NMR relaxation to demonstrate the presence of a solvent-driven dynamical crossover between different motional regimes, often also referred to as the dynamical transition. In particular, the dynamics are dominated by small-angle fluctuations at low temperatures and by ?-flips of the aromatic ring at high temperatures. The crossover temperature is more than 43 degrees lower for the hydrated state of the fibrils compared to the dry state, indicating that interactions with water facilitate ?-flips. Further, crossover temperatures are shown to be very sensitive to polymorphic states of the fibrils, such as the 2-fold and 3-fold symmetric morphologies of the wild-type protein as well as D23N mutant protofibrils. We speculate that these differences can be attributed, at least partially, to enhanced interactions with water in the 3-fold polymorph, which has been shown to have a water-accessible cavity. Combined with previous studies of methyl group dynamics, the results highlight the presence of multiple dynamics modes in the core of the fibrils, which was originally believed to be quite rigid.
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Project description:We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7?min) to 20?% piperidine at pH?11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH?7.
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Project description:Serine side-chains are strategic sites of post-translational modifications, and it is important to establish benchmarks of their internal dynamics. In this work, we compare the dynamics of serine side-chains in several biologically important systems: serine-8 in the disordered domain of Aβ<sub>1-40</sub> fibrils in the hydrated and dry states and fluorenylmethyloxycarbonyl (Fmoc) serine with the bulky group that mimics the hydrophobicity of the fibril contacts yet lacks the complexity of the protein system. Using deuterium solid-state NMR static line shape and longitudinal relaxation techniques in the 310 to 180 K temperature range, we compare the main features of the dynamics in these systems. The main motional modes in the fibrils are large-scale fluctuations in the hydrated state of the fibrils as well as local motions such as 3-site jumps of the C<sup>β</sup> deuterons at high temperatures and small-angle fluctuations of the C<sup>α</sup>-C<sup>β</sup> axis at low temperatures. In the hydrated fibrils, two distinct states are present with vastly different extents of large-scale diffusive motions and 3-site-jump rate constants. The hydrated state at the physiological conditions is dominated by the "free" state undergoing large-scale diffusive motions and very fast local 3-site jumps, while in the "bound" state, these large-scale motions are quenched due to transient inter- and intramolecular interactions. Additionally, in the bound state, the 3-site-jump motions are orders of magnitude slower. Details of the dynamics in the serine side-chain are dependent on fine structural features and hydration levels of the systems.
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Project description:Using all-atom explicit solvent replica exchange molecular dynamics simulations with solute tempering, we study the effect of methionine oxidation on A?10-40 peptide binding to the zwitterionic DMPC bilayer. By comparing oxidized and reduced peptides, we identified changes in the binding mechanism caused by this modification. First, Met35 oxidation unravels C-terminal helix in the bound peptides. Second, oxidation destabilizes intrapeptide interactions and expands bound peptides. We explain these outcomes by the loss of amphiphilic character of the C-terminal helix due to oxidation. Third, oxidation "polarizes" A? binding to the DMPC bilayer by strengthening the interactions of the C-terminus with lipids while largely releasing the rest of the peptide from bilayer. Fourth, in contrast to the wild-type peptide, oxidized A? induces significantly smaller bilayer thinning and drop in lipid density within the binding footprint. These observations are the consequence of mixing oxidized peptide amino acids with lipids promoted by enhanced A? conformational fluctuations. Fifth, methionine oxidation reduces the affinity of A? binding to the DMPC bilayer by disrupting favorable intrapeptide interactions upon binding, which offset the gains from better hydration. Reduced binding affinity of the oxidized A? may represent the molecular basis for its reduced cytotoxicity.
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Project description:A solution-phase automated synthesis of the versatile synthetic intermediate, Garner's aldehyde, was demonstrated. <i>tert</i>-Butoxycarbonyl (Boc) protection, acetal formation, and reduction of the ester to the corresponding aldehyde were performed utilizing our originally developed automated synthesizer, ChemKonzert. The developed procedure was also useful for the synthesis of Garner's aldehyde analogues possessing fluorenylmethyloxycarbonyl (Fmoc) or benzyloxycarbonyl (Cbz) protection.
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Project description:Mammalian cells require activated folates to generate nucleotides for growth and division. The most abundant circulating folate species is 5-methyl tetrahydrofolate (5-methyl-THF), which is used to synthesize methionine from homocysteine via the cobalamin-dependent enzyme methionine synthase (MTR). Cobalamin deficiency traps folates as 5-methyl-THF. Here, we show using isotope tracing that MTR is only a minor source of methionine in cell culture, tissues or xenografted tumours. Instead, MTR is required for cells to avoid folate trapping and assimilate 5-methyl-THF into other folate species. Under conditions of physiological extracellular folates, genetic MTR knockout in tumour cells leads to folate trapping, purine synthesis stalling, nucleotide depletion and impaired growth in cell culture and as xenografts. These defects are rescued by free folate but not one-carbon unit supplementation. Thus, MTR plays a crucial role in liberating THF for use in one-carbon metabolism.
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Project description:Although most organisms synthesize methionine from homocysteine and methyl folates, some have "core" methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.
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