6,050 research outputs found
Modular Use of the Uniquely Small Ring A of Mersacidin Generates the Smallest Ribosomally Produced Lanthipeptide
Mersacidin is an antimicrobial class II lanthipeptide. Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by intramolecular lanthionine rings. These rings give lanthipeptides their bioactive structure and stability. RiPPs are produced from a gene cluster that encodes a precursor peptide and its dedicated unique modification enzymes. The field of RiPP engineering aims to recombine modification enzymes from different RiPPs to modify new substrates, resulting in new-to-nature molecules with novel or improved functionality. The enzyme MrsM from the mersacidin gene cluster installs the four lanthionine rings of mersacidin, including the uniquely small ring A. By applying MrsM in RiPP engineering, this ring could be installed in linear peptides to achieve stabilization by a very small lanthionine or to create small lanthionine-stabilized modules for chemical modification. However, the formation of unique intramolecular structures like that of mersacidin's ring A can be very stringent. Here, the formation of ring A of mersacidin is characterized by mutagenesis. A range of truncated mersacidin variants was made to identify the smallest possible construct in which this ring could still be formed. Additionally, mutants were created to study the flexibility of ring A formation. It was found that although the formation of ring A is stringent, it can be formed in a core peptide as small as five amino acids. The truncated mersacidin core peptide CTFAL is the smallest ribosomally produced lanthipeptide reported to date, and it has exciting prospects as a new module for application in RiPP engineering
Two-vibron bound states in alpha-helix proteins : the interplay between the intramolecular anharmonicity and the strong vibron-phonon coupling
The influence of the intramolecular anharmonicity and the strong
vibron-phonon coupling on the two-vibron dynamics in an -helix protein
is studied within a modified Davydov model. The intramolecular anharmonicity of
each amide-I vibration is considered and the vibron dynamics is described
according to the small polaron approach. A unitary transformation is performed
to remove the intramolecular anharmonicity and a modified Lang-Firsov
transformation is applied to renormalize the vibron-phonon interaction. Then, a
mean field procedure is realized to obtain the dressed anharmonic vibron
Hamiltonian. It is shown that the anharmonicity modifies the vibron-phonon
interaction which results in an enhancement of the dressing effect. In
addition, both the anharmonicity and the dressing favor the occurrence of two
different bound states which the properties strongly depend on the interplay
between the anharmonicity and the dressing. Such a dependence was summarized in
a phase diagram which characterizes the number and the nature of the bound
states as a function of the relevant parameters of the problem. For a
significant anharmonicity, the low frequency bound states describe two vibrons
trapped onto the same amide-I vibration whereas the high frequency bound states
refer to the trapping of the two vibrons onto nearest neighbor amide-I
vibrations.Comment: may 2003 submitted to Phys. Rev.
Classical and quantum LTB model for the non-marginal case
We extend the classical and quantum treatment of the Lemaitre-Tolman-Bondi
(LTB) model to the non-marginal case (defined by the fact that the shells of
the dust cloud start with a non-vanishing velocity at infinity). We present the
classical canonical formalism and address with particular care the boundary
terms in the action. We give the general relation between dust time and Killing
time. Employing a lattice regularization, we then derive and discuss for
particular factor orderings exact solutions to all quantum constraints.Comment: 23 pages, no figures, typos correcte
Synthesis and Characterization of Heterodimers and Fluorescent Nisin Species by Incorporation of Methionine Analogues and Subsequent Click Chemistry
Noncanonical amino acids form a highly diverse pool of building blocks that can render unique physicochemical properties to peptides and proteins. Here, four methionine analogues with unsaturated and varying side chain lengths were successfully incorporated at four different positions in nisin in Lactococcus lactis through force feeding. This approach allows for residue-specific incorporation of methionine analogues into nisin to expand their structural diversity and alter their activity profiles. Moreover, the insertion of methionine analogues with biorthogonal chemical reactivity, e.g., azidohomoalanine and homopropargylglycine, provides the opportunity for chemical coupling to functional moieties and fluorescent probes as well as for intermolecular coupling of nisin variants. All resulting nisin conjugates retained antimicrobial activity, which substantiates the potential of this method as a tool to further study its localization and mode of action
Semisynthetic Macrocyclic Lipo-lanthipeptides Display Antimicrobial Activity Against Bacterial Pathogens
[Image: see text] A large number of antimicrobial peptides depend on intramolecular disulfide bonds for their biological activity. However, the relative instability of disulfide bonds has limited the potential of some of these peptides to be developed into therapeutics. Conversely, peptides containing intramolecular (methyl)lanthionine-based bonds, lanthipeptides, are highly stable under a broader range of biological and physical conditions. Here, the class-II lanthipeptide synthetase CinM, from the cinnamycin gene cluster, was employed to create methyllanthionine stabilized analogues of disulfide-bond-containing antimicrobial peptides. The resulting analogues were subsequently modified in vitro by adding lipid tails of variable lengths through chemical addition. Finally, the created compounds were characterized by MIC tests against several relevant pathogens, killing assays, membrane permeability assays, and hemolysis assays. It was found that CinM could successfully install methyllanthionine bonds at the intended positions of the analogues and that the lipidated macrocyclic core peptides have bactericidal activity against tested Gram-positive and Gram-negative pathogenic bacteria. Additionally, fluorescence microscopy assays revealed that the lipidated compounds disrupt the bacterial membrane and lyse bacterial cells, hinting toward a potential mode of action. Notably, the semisynthesized macrocyclic lipo-lanthipeptides show low hemolytic activity. These results show that the methods developed here extend the toolbox for novel antimicrobial development and might enable the further development of novel compounds with killing activity against relevant pathogenic bacteria
Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography
<p>Abstract</p> <p>Background</p> <p>During the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells <it>in vitro </it>and <it>in vivo</it>. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.</p> <p>Methods</p> <p>Here we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.</p> <p>Results</p> <p>Our results show that unconcentrated LV vector stocks with titers in excess of 10<sup>8 </sup>transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm<sup>2 </sup>tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 Ă— 10<sup>10 </sup>TU were recovered from a single HYPERFlask vessel.</p> <p>Conclusion</p> <p>The protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.</p
Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export
The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing
Conformational States of Melittin at a Bilayer Interface
AbstractThe distribution of peptide conformations in the membrane interface is central to partitioning energetics. Molecular-dynamics simulations enable characterization of in-membrane structural dynamics. Here, we describe melittin partitioning into dioleoylphosphatidylcholine lipids using CHARMM and OPLS force fields. Although the OPLS simulation failed to reproduce experimental results, the CHARMM simulation reported was consistent with experiments. The CHARMM simulation showed melittin to be represented by a narrow distribution of folding states in the membrane interface
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