scholarly journals Biomimetic Elastin-Like Polypeptides as Materials for the Activation of Mechanophoric Catalysts

2020 ◽  
Vol 02 (02) ◽  
pp. 116-128 ◽  
Author(s):  
Sebastian Funtan ◽  
Anne Funtan ◽  
Reinhard Paschke ◽  
Wolfgang H. Binder
Keyword(s):  
Mechanochemical Activation ◽  
Click Reaction ◽  
Mechanical Energy ◽  
Cd Spectroscopy ◽  
Applied Force ◽  
Type Ii ◽  
Secondary Structure Analysis ◽  
Efficient Uptake ◽  
Molecular Springs ◽  
Chain Lengths

Elastin-like polypeptides (ELPs) are well known for their elastic and thermoresponsive behaviors. Their elasticity originates from the formation of a β-spiral which is the consequence of stacking type-II β-turns, formed from individual VPGVG pentapeptide units. Here, the synthesis of ELPs of varying chain lengths [VPGVG, (VPGVG)2, and (VPGVG)4] and their coupling to a mechanoresponsive catalyst are reported. The attached ELP chains can act as “molecular springs,” allowing for an efficient uptake and transmission of an applied force to the mechanophoric bond. This leads to stress-induced activation of the mechanophoric catalyst, in turn transforming mechanical energy into a “click” reaction. Secondary structure analysis via IR and CD spectroscopy revealed that the β–spiral formation of the ELP is not affected by the coupling process and the β–spiral is still intact in the mechanocatalyst after the coupling. Mechanochemical activation of the synthesized catalysts by an external applied force, studied via ultrasonication, showed conversions of the copper(I)-catalyzed alkyne-azide “click” reaction (CuAAC) up to 5.6% with an increasing chain length of the peptide, proving the potential to incorporate this chemistry into biomaterial engineering.

scholarly journals BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy

2020 ◽  
pp. 175-189
Author(s):  
András Micsonai ◽  
Éva Bulyáki ◽  
József Kardos
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Classical Method ◽  
Cd Spectroscopy ◽  
Protein Fold ◽  
Cd Spectra ◽  
Secondary Structure Analysis ◽  
Β Sheets ◽  
Α Helix ◽  
Circular Dichroïsm

Abstract Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold. The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.

scholarly journals PSVI-20 The Effect of Resistant Starch Type II on Satiety Hormones of Fasting Dogs

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 381-382
Author(s):  
Isabella Corsato Alvarenga ◽  
Dennis E Jewell ◽  
Matthew Jackson ◽  
Charles G Aldrich
Keyword(s):  
Resistant Starch ◽  
Mixed Model ◽  
Peptide Yy ◽  
Mechanical Energy ◽  
Latin Square ◽  
Gastric Inhibitory Polypeptide ◽  
Blood Stream ◽  
Future Research ◽  
Multiple Time ◽  
Type Ii

Abstract Resistant starches comprise the starch fraction that escapes small intestine (SI) digestion and reaches the colon, where it is fermented by saccharolytic bacteria that produce beneficial postbiotics. Moreover, slowly digestible starches lower glycemic index and may promote satiety. The objectives of this study were to assess whether diets having increased RS perturbed fasting levels of satiety hormones in dogs. Diets with three levels of resistant starch (RS type II) were produced via extrusion at three levels of mechanical energy. Nine male and 15 female adult Beagle dogs were fed the low, medium and high RS foods in a 3x3 Latin square design (n = 24). The study was approved by IACUC at Hill’s Pet Nutrition (# 883.0.0.0). Fasting blood was collected in the morning on days 27 and 28 of each period, and serum was separated and frozen at -70°C until analysis. Satiety hormones were measured by ELISA. Data was first converted to natural log to approximate normal distribution and then analyzed as a mixed model by the GLIMMIX procedure from SAS (v 9.4) with diet as fixed effect and dog and period as random effects. Ghrelin, gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP-1), glucagon, leptin, pancreatic polypeptide (PP) and peptide YY (PYY) were not different across treatments likely due to the fasting state of dogs. Surprisingly, only insulin increased with higher RS consumption. It is possible that more propionate (not measured) was produced in the colon of dogs fed the high RS diet, which went through gluconeogenesis in the liver and resulted in a change in the time of glucose entering the blood stream. This could explain the insulin increase during fasting, but needs to be verified by measurement of fecal SCFA. Future research should explore the effect of type II RS on satiety hormones at multiple time points postprandial.

scholarly journals Tissue mechanics govern the rapidly adapting and symmetrical response to touch

2015 ◽  
Vol 112 (50) ◽  
pp. E6955-E6963 ◽  
Author(s):  
Amy L. Eastwood ◽  
Alessandro Sanzeni ◽  
Bryan C. Petzold ◽  
Sung-Jin Park ◽  
Massimo Vergassola ◽  
...  
Keyword(s):  
Mechanical Energy ◽  
Physical World ◽  
Tissue Mechanics ◽  
Applied Force ◽  
General Mechanism ◽  
Channel Activation ◽  
Mechanoelectrical Transduction ◽  
Mechanical Filter ◽  
Somatosensory Neurons

Interactions with the physical world are deeply rooted in our sense of touch and depend on ensembles of somatosensory neurons that invade and innervate the skin. Somatosensory neurons convert the mechanical energy delivered in each touch into excitatory membrane currents carried by mechanoelectrical transduction (MeT) channels. Pacinian corpuscles in mammals and touch receptor neurons (TRNs) in Caenorhabditis elegans nematodes are embedded in distinctive specialized accessory structures, have low thresholds for activation, and adapt rapidly to the application and removal of mechanical loads. Recently, many of the protein partners that form native MeT channels in these and other somatosensory neurons have been identified. However, the biophysical mechanism of symmetric responses to the onset and offset of mechanical stimulation has eluded understanding for decades. Moreover, it is not known whether applied force or the resulting indentation activate MeT channels. Here, we introduce a system for simultaneously recording membrane current, applied force, and the resulting indentation in living C. elegans (Feedback-controlled Application of mechanical Loads Combined with in vivo Neurophysiology, FALCON) and use it, together with modeling, to study these questions. We show that current amplitude increases with indentation, not force, and that fast stimuli evoke larger currents than slower stimuli producing the same or smaller indentation. A model linking body indentation to MeT channel activation through an embedded viscoelastic element reproduces the experimental findings, predicts that the TRNs function as a band-pass mechanical filter, and provides a general mechanism for symmetrical and rapidly adapting MeT channel activation relevant to somatosensory neurons across phyla and submodalities.

scholarly journals Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1

1998 ◽  
Vol 335 (2) ◽  
pp. 375-380 ◽  
Author(s):  
Stephen P. BRAZIER ◽  
Bala. RAMESH ◽  
Parvez I. HARIS ◽  
David C. LEE ◽  
Surjit K. S. SRAI
Keyword(s):  
Gradient Centrifugation ◽  
Helical Structure ◽  
Structural Data ◽  
Cd Spectroscopy ◽  
Inward Rectifier ◽  
K Channel ◽  
Phospholipid Membranes ◽  
K Channels ◽  
Voltage Gated ◽  
Secondary Structure Analysis

The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a ‘pore ’ (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85–96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an α-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255–262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular β-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an α-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an α-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.

scholarly journals The Hirudo Medicinalis Microbiome Is a Source of New Antimicrobial Peptides

2020 ◽  
Vol 21 (19) ◽  
pp. 7141
Author(s):  
Ekaterina Grafskaia ◽  
Elizaveta Pavlova ◽  
Vladislav V. Babenko ◽  
Ivan Latsis ◽  
Maja Malakhova ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Cd Spectroscopy ◽  
Hirudo Medicinalis ◽  
Analysis Method ◽  
Promising Candidate ◽  
New Class ◽  
Disordered Structures ◽  
Secondary Structure Analysis ◽  
Helical Content ◽  
Strong Antimicrobial Activity

Antimicrobial peptides (AMPs) are considered a promising new class of anti-infectious agents. This study reports new antimicrobial peptides derived from the Hirudo medicinalis microbiome identified by a computational analysis method applied to the H. medicinalis metagenome. The identified AMPs possess a strong antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC range: 5.3 to 22.4 μM), including Staphylococcus haemolyticus, an opportunistic coagulase–negative pathogen. The secondary structure analysis of peptides via CD spectroscopy showed that all the AMPs except pept_352 have mostly disordered structures that do not change under different conditions. For peptide pept_352, the α–helical content increases in the membrane environment. The examination of the mechanism of action of peptides suggests that peptide pept_352 exhibits a direct membranolytic activity. Furthermore, the cytotoxicity assay demonstrated that the nontoxic peptide pept_1545 is a promising candidate for drug development. Overall, the analysis method implemented in the study may serve as an effective tool for the identification of new AMPs.

scholarly journals Extraction and characterization of the tissue forms of collagen types II and IX from bovine vitreous

1994 ◽  
Vol 299 (2) ◽  
pp. 497-505 ◽  
Author(s):  
P N Bishop ◽  
M V Crossman ◽  
D McLeod ◽  
S Ayad
Keyword(s):  
Chondroitin Sulphate ◽  
Minor Component ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Collagen Types ◽  
The Matrix ◽  
Type V ◽  
Chain Lengths ◽  
First Time

We report for the first time that, after centrifugation of adult bovine vitreous, the hyaluronan-rich supernatant contains collagens which can be isolated in their intact forms by precipitation with 4.5 M NaCl. This precipitate constituted approx. 4% of the total vitreous collagen and comprised collagen types IX and II (in the approximate ratio of 4:1) with negligible amounts of type-V/XI collagen. Type-II collagen was present partly in a pro-alpha 1(II) form, suggesting that there is active synthesis of type-II collagen into the matrix of adult bovine vitreous. Type-IX collagen was purified (2-2.5 mg/l of vitreous) and its glycosaminoglycan chain composition was analysed. Bovine vitreous type-IX collagen always possessed a glycosaminoglycan chain of comparatively low M(r) that was predominantly 4-sulphated, with chondroitin 6-sulphate representing a more minor component. By contrast, chick vitreous has been shown to contain type-IX collagen which always possesses a high-M(r) chondroitin sulphate chain that is predominantly 6-sulphated. The functional significance of these different glycosaminoglycan chain lengths and sulphation patterns is discussed.

scholarly journals Synthesis and investigation of quadruplex-DNA-binding, 9-O-substituted berberine derivatives

2020 ◽  
Vol 16 ◽  
pp. 2795-2806
Author(s):  
Jonas Becher ◽  
Daria V Berdnikova ◽  
Heiko Ihmels ◽  
Christopher Stremmel
Keyword(s):  
Binding Affinity ◽  
Click Reaction ◽  
Thermal Stabilization ◽  
Cd Spectroscopy ◽  
Fluorescent Light ◽  
Quadruplex Dna ◽  
G4 Dna ◽  
Small Series ◽  
Berberine Derivatives ◽  
Dna Complex

A small series of five novel berberine derivatives was synthesized by the Cu-catalyzed click reaction of 9-propargyladenine with 9-O-(azidoalkyl)berberine derivatives. The association of the resulting berberine–adenine conjugates with representative quadruplex-forming oligonucleotides 22AG dA(G3TTA)3G3 and a2 d(ACAG4TGTG4)2 was examined with photometric and fluorimetric titrations, thermal DNA denaturation analysis, and CD spectroscopy. The results from the spectrometric titrations indicated the formation of 2:1 or 1:1 complexes (ligand:G4-DNA) with log K b values of 10–11 (2:1) and 5–6 (1:1), which are typical for berberine derivatives. Notably, a clear relationship between the binding affinity of the ligands with the length of the alkyl linker chain, n, was not observed. However, depending on the structure, the ligands exhibited different effects when bound to the G4-DNA, such as fluorescent light-up effects and formation of ICD bands, which are mostly pronounced with a linker length of n = 4 (with a2) and n = 5 (with 22AG), thus indicating that each ligand–G4-DNA complex has a specific structure with respect to relative alignment and conformational flexibility of the ligand in the binding site. It was shown exemplarily with one representative ligand from the series that such berberine–adenine conjugates exhibit a selective binding, specifically a selectivity to quadruplex DNA in competition with duplex DNA, and a preferential thermal stabilization of the G4-DNA forms 22AG and KRAS. Notably, the experimental data do not provide evidence for a significant effect of the adenine unit on the binding affinity of the ligands, for example, by additional association with the loops, presumably because the adenine residue is sterically shielded by the neighboring triazole unit.

scholarly journals Synthesis and Mechanochemical Activity of Peptide-Based Cu(I) Bis(N-Heterocyclic Carbene) Complexes

Biomimetics ◽  
2019 ◽  
Vol 4 (1) ◽  
pp. 24 ◽  
Author(s):  
Sebastian Funtan ◽  
Philipp Michael ◽  
Wolfgang H. Binder
Keyword(s):  
Click Reaction ◽  
Mechanical Energy ◽  
Reaction Force ◽  
Interesting Property ◽  
Mechanical Stimuli ◽  
Force Transmission ◽  
Carbene Complexes ◽  
C Terminus ◽  
Post Modification ◽  
Chemical Response

With the class of shock-absorbing proteins, nature created some of the most robust materials combining both mechanical strength and elasticity. Their excellent ability to dissipate energy to prevent surrounding cells from damage is an interesting property that regularly is exploited for applications in biomimetic materials. Similar to biomaterials, where mechanical stimuli are transmitted into a (bio)chemical response, mechanophoric catalysts transform mechanical energy into a chemical reaction. Force transmission is realized commonly by polymeric handles directing the applied force to the mechanophoric bond, which in turn leads to stress-induced activation of the catalyst. Therefore, shock-absorbing proteins able to take up and store mechanical energy elastically for subsequent force transduction to the labile bond seem to be perfect candidates to fulfill this task. Here, we report on the synthesis of two different latent mechanophoric copper(I) bis(N-heterocyclic carbene) complexes bearing either two carboxyl groups or two amino groups which allow conjugation reactions with either the N- or the C-terminus of amino acids or peptides. The chosen catalysts can be activated, for instance, by applying external mechanical force via ultrasound, removing one N-heterocyclic carbene (NHC) ligand. Post-modification of the mechanophoric catalysts via peptide coupling (Gly, Val) and first reactions showed that the mechanoresponsive behavior was still present after the coupling. Subsequent polycondensation of both catalysts lead to a polyamide including the Cu(I) moiety. Mechanochemical activation by ultrasound showed conversions in the copper(I)-catalyzed alkyne-azide “click” reaction (CuAAC) up to 9.9% proving the potential application for the time and spatial controlled CuAAC.

scholarly journals Terminal Mono- and Bis-Conjugates of Oligonucleotides with Closo-Dodecaborate: Synthesis and Physico-Chemical Properties

2020 ◽  
Author(s):  
Darya Novopashina ◽  
Mariya A. Vorobyeva ◽  
Alexander A. Lomzov ◽  
Vladimir N. Silnikov ◽  
Alya G. Venyaminova
Keyword(s):  
Molecular Biology ◽  
Structural Changes ◽  
Click Reaction ◽  
Double Helix ◽  
Chemical Properties ◽  
Cd Spectroscopy ◽  
Boron Clusters ◽  
Strong Stabilization ◽  
Duplex Stability ◽  
Oligonucleotide Conjugates

Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, closo-dodecaborate anion represents a high-boron containing residue with remarkable chemical stability and low toxicity, suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, while analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized DNA/DNA duplex but gave a strong stabilization of the duplex with RNA target. According to CD spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.

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