Atomization mechanism
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Even though amyloid aggregates were discovered many years ago the mechanism of their formation is still a mystery. Because of their connection to many of untreatable neurodegenerative diseases the motivation for finding a common aggregation path is high. We report a new high heat induced fibrillization path of a model protein β-lactoglobulin (BLG) when incubated in glycine instead of water at pH 2. By combining atomic force microscopy (AFM), transmission emission microscopy (TEM), dynamic light scattering (DLS) and circular dichroism (CD) we predict that the basic building blocks of fibrils made in glycine are not peptides, but rather spheroid oligomers of different height that form by stacking of ring-like structures. Spheroid oligomers linearly align to form fibrils by opening up and combining. We suspect that glycine acts as an hydrolysation inhibitor which consequently promotes a different fibrillization path. By combining the known data on fibrillization in water with our experimental conclusions we come up with a new fibrillization scheme for BLG. We show that by changing the fibrillization conditions just by small changes in buffer composition can dramatically change the aggregation pathway and the effect of buffer shouldn't be neglected. Fibrils seen in our study are also gaining more and more attention because of their pore-like structure and a possible cytotoxic mechanism by forming pernicious ion-channels. By preparing them in a simple model system as BLG we opened a new way to study their formation.
- MeSH
- amyloid * chemie MeSH
- glycin farmakologie MeSH
- laktoglobuliny * chemie MeSH
- mikroskopie atomárních sil metody MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
Hypothesis of coherent vibration states in biological systems based on nonlinear interaction between longitudinal elastic and electric polarization fields with metabolic energy supply was formulated by Frohlich. Conditions for excitation of coherent states and generation of electromagnetic fields are satisfied in microtubules which form electrical polar structures. Numerical models are used for analysis of Frohlich's vibration states in cells. Reduction of activity and of energy production in mitochondria, and disintegration of cytoskeleton structures by phosphorylation on the pathway of cancer trasformation can diminish excitation of the Frohlich's vibration states and of the generated electromagnetic field, which results in disturbances of the interaction forces between cells. Interaction forces between cancer cells may be smaller than interaction forces between healthy cells and cancer cells as follows from numerical models. Mechanism of malignity, i.e. local invasion, detachment of cancer cells, and metastasis, is assumed to depend on the electromagnetic field.
- MeSH
- biofyzika metody MeSH
- biologické modely MeSH
- buňky 3T3 MeSH
- cytoskelet metabolismus MeSH
- elektromagnetická pole MeSH
- elektromagnetické jevy MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- mikrotubuly metabolismus MeSH
- mitochondrie metabolismus MeSH
- myši MeSH
- nádory metabolismus MeSH
- pružnost MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Adsorpcí biopolymerů na povrch titanu a titanem dopovaných uhlovodíkových povlaků se zvyšuje optická drsnost, tloušťka a index lomu povrchové vrstvy. Fibrinogen se lépe adsorbuje na povrchu titanu, který je upraven leštěním a leptáním, nežli na povrchu pouze leštěném. Nejlépe se adsorbuje na povrch uhlovodíkových povlaků dopovaným titanem, které byly deponované reaktivním magnetronovým naprašováním v optimálním poměru Ti0,38 - C0,62 a Ti0,09 - C0,91. Takto upravené povrchy dentálních implantátů by měly urychlit jejich oseointegraci a vhojení. Na povrch titanu se lépe adsorbují jednořetězové pyrimidinové oligodeoxynukleotidy (TTC)12, nežli purinové (AAG)12. Dvojšroubovicový duplex (TTC)12. (AAG)12 neovlivňuje optické vlastnosti povrchu titanu a patrně se na povrch neadsorbuje.
Adsorption of biopolymers at the titanium and titanium carbide surface increases the optical roughness as well as the thickness and refractive index of the surface layer. Fibrinogen is better adsorbed at the titanium surface which is treated by polishing and etching than at the surface treated only by polishing. The best adsorption of fibrinogen was observed at the titanium carbide surface prepared by plasma-enhanced chemical vapour deposition, the optimal ratio was Ti0,38 - C0,62 a Ti0,09 - C0,91. The surface of dental implants treated by this way should speed up their osseointegration and healing. The single stranded pyrimidine oligodeoxynucleotides (TTC)12 are better adsorbed at the titanium surface than the purine oligodeoxynucleotides (AAG)12. The double-helical duplex (TTC)12. (AAG)12 has no effect on the optical properties of the titanium surface and probably is not adsorbed there.
- MeSH
- adsorpce MeSH
- biokompatibilní materiály normy MeSH
- biosenzitivní techniky využití MeSH
- fibrinogen MeSH
- financování organizované MeSH
- mikroskopie atomárních sil využití MeSH
- oligonukleotidy MeSH
- osteoblasty ultrastruktura MeSH
- osteointegrace fyziologie MeSH
- techniky in vitro MeSH
- titan terapeutické užití MeSH
- zubní implantáty MeSH
In this work we evaluate the applicability of different atomic force microscopy (AFM) modes, such as Phase Shift Imaging, Atomic Force Acoustic Microscopy (AFAM) and Force Spectroscopy, for mapping of the distribution pattern of low-molecular-weight biomimetic groups on polymer biomaterial surfaces. Patterns with either random or clustered spatial distribution of bioactive peptide group derived from fibronectin were prepared by surface deposition of functional block copolymer nano-colloids and grafted with RGDS peptide containing the sequence of amino acids arginine-glycine-aspartic acid-serine (conventionally labeled as RGDS) and carrying biotin as a tag. The biotin-tagged peptides were labeled with 40nm streptavidin-modified Au nanospheres. The peptide molecules were localized through the detection of bound Au nanospheres by AFM, and thus, the surface distribution of peptides was revealed. AFM techniques capable of monitoring local mechanical properties of the surface were proved to be the most efficient for identification of Au nano-markers. The efficiency was successfully demonstrated on two different patterns, i.e. random and clustered distribution of RGDS peptides on structured surface of the polymer biomaterial.
Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs' functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains.
The RNA-dependent RNA polymerases of Flaviviridae viruses are crucial for replication. The Flaviviridae polymerase is organized into structural motifs (A-G), with motifs F, A, C and E containing interrogating, priming and catalytic substrate-interacting sites. Modified nucleoside analogues act as antiviral drugs by targeting Flaviviridae polymerases and integrating into the synthesized product causing premature termination. A threonine mutation of a conserved serine residue in motif B of Flaviviridae polymerases renders resistance to 2'-C-methylated nucleoside analogues. The mechanism how this single mutation causes Flaviviridae viruses to escape nucleoside analogues is not yet known. Given the pivotal position of the serine residue in motif B that supports motif F, we hypothesized the threonine mutation causes alterations in nucleoside exploration within the entry tunnel. Implementing a stochastic molecular software showed the all-atom 2'-C-methylated analogue reaction within the active sites of wild type and serine-threonine mutant polymerases from Hepacivirus and Flavivirus. Compared with the wild type, the serine-threonine mutant polymerases caused a significant decrease of analogue contacts with conserved interrogating residues in motif F and a displacement of metal ion cofactors. The simulations significantly showed that during the analogue exploration of the active site the hydrophobic methyl group in the serine-threonine mutant repels water-mediated hydrogen bonds with the 2'-C-methylated analogue, causing a concentration of water-mediated bonds at the substrate-interacting sites. Collectively, the data are an insight into a molecular escape mechanism by Flaviviridae viruses from 2'-C-methylated nucleoside analogue inhibitors.
The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.
- MeSH
- analýza dat MeSH
- elektrofyziologické jevy * MeSH
- kardiomyocyty cytologie fyziologie MeSH
- mechanické jevy * MeSH
- mikroskopie atomárních sil * přístrojové vybavení metody MeSH
- molekulární zobrazování MeSH
- spřažení excitace a kontrakce * MeSH
- vápníková signalizace MeSH
- Publikační typ
- časopisecké články MeSH
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 A resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be DeltaG(++) approximately 22(+/-5) kcal x mol(-1), which is in a good agreement with the experimentally observed reaction rate constant (k(cat) approximately 1 s(-1)). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.
- MeSH
- alanin metabolismus MeSH
- biologické modely MeSH
- dipeptidy genetika metabolismus MeSH
- financování organizované MeSH
- glutamátkarboxypeptidasa II genetika chemie metabolismus MeSH
- hydrolýza MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutageneze MeSH
- substituce aminokyselin MeSH
- substrátová specifita genetika MeSH
- termodynamika MeSH
- vazba proteinů genetika MeSH
- vazebná místa genetika MeSH
- vodíková vazba MeSH
- výpočetní biologie metody MeSH
- zinek chemie MeSH
- Check Tag
- lidé MeSH
The S-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ from Streptomyces caelestis catalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0 Å resolution, its native form at 2.7 Å resolution and its complex with S-adenosyl-L-homocysteine (SAH) at 2.9 Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ-SAM-substrate and CcbJ-SAH-product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H(+) ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.
A detailed study of the biological effects of diverse quality radiations, addressing their biophysical interpretation, is presented. Published survival data for V79 cells irradiated by monoenergetic protons, helium-3, carbon and oxygen ions and for CHO cells irradiated by carbon ions have been analysed using the probabilistic two-stage model of cell inactivation. Three different classes of DNA damage formed by traversing particles have been distinguished, namely severe single-track lesions which might lead to cell inactivation directly, less severe lesions where cell inactivation is caused by their combinations and lesions of negligible severity that can be repaired easily. Probabilities of single ions forming these lesions have been assessed in dependence on their linear energy transfer (LET) values. Damage induction probabilities increase with atomic number and LET. While combined lesions play a crucial role at lower LET values, single-track damage dominates in high-LET regions. The yields of single-track lethal lesions for protons have been compared with Monte Carlo estimates of complex DNA lesions, indicating that lethal events correlate well with complex DNA double-strand breaks. The decrease in the single-track damage probability for protons of LET above approximately 30 keV microm(-1), suggested by limited experimental evidence, is discussed, together with the consequent differences in the mechanisms of biological effects between protons and heavier ions. Applications of the results in hadrontherapy treatment planning are outlined.
- MeSH
- biologické modely MeSH
- buněčné linie MeSH
- financování organizované MeSH
- ionty MeSH
- křečci praví MeSH
- kyslík chemie MeSH
- lineární přenos energie genetika MeSH
- metoda Monte Carlo MeSH
- oprava DNA genetika MeSH
- poškození DNA fyziologie účinky záření MeSH
- protony MeSH
- uhlík chemie MeSH
- viabilita buněk fyziologie účinky záření MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
