Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are ...either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs.
Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied
in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers
in vivo.
•The conjugation of chitosan of different sizes on DNA compaction and particle formation is studied here.•Chitosan binds DNA via electrostatic and hydrophilic interactions.•Chitosan-100 forms ...stronger DNA conjugate than chitosan-200 ad chitosan-15kD.•Major DNA compaction and particle formation occurred with chitosan-100 and chitosan-200kD.•A partial B to A-DNA transition was observed in the presence of chitosan-100 and chitosan-200kD.
Conjugations of DNA with chitosans 15kD (ch-15), 100kD (ch-100) and 200kD (ch-200) were investigated in aqueous solution at pH 5.5–6.5. Multiple spectroscopic methods and atomic force microscopy (AFM) were used to locate the chitosan binding sites and the effect of polymer conjugation on DNA compaction and particle formation. Structural analysis showed that chitosan–DNA conjugation is mainly via electrostatic interactions through polymer cationic charged NH2 and negatively charged backbone phosphate groups. As polymer size increases major DNA compaction and particle formation occurs. At high chitosan concentration major DNA structural changes observed indicating a partial B to A-DNA conformational transition.
Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape. Among dendrimers, polyamidoamine (PAMAM) have received most attention as ...potential transfection agents for gene delivery, because these macromolecules bind DNA at physiological pH. The aim of this study was to examine the interaction of calf-thymus DNA with several dendrimers of different compositions, such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant DNA concentration and various dendrimer contents. FTIR, UV−visible, and CD spectroscopic methods, as well as atomic force microscopy (AFM), were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on DNA stability, aggregation, condensation, and conformation. Structural analysis showed a strong dendrimer−DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K mPEG-G3 = 1.5 (±0.5) × 103 M−1, K mPEG-G4 = 3.4 (±0.80) × 103 M−1, and K PAMAM-G4 = 8.2 (±0.90) × 104 M−1. The order of stability of polymer−DNA complexation is PAMAM-G4 > mPEG-G4 > mPEG-G3. Both hydrophilic and hydrophobic interactions were observed for dendrimer−DNA complexes. DNA remained in the B-family structure, while biopolymer particle formation and condensation occurred at high dendrimer concentrations.
Mutations in the intermediate filament (IF) protein desmin cause severe forms of myofibrillar myopathy characterized by partial aggregation of the extrasarcomeric desmin cytoskeleton and structural ...disorganization of myofibrils. In contrast to prior expectations, we showed that some of the known disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are assembly-competent and do allow formation of
bona fide IFs
in vitro and
in vivo. We also previously demonstrated that atomic force microscopy can be employed to measure the tensile properties of single desmin IFs. Using the same approach on filaments formed by the aforementioned mutant desmins, we now observed two different nanomechanical behaviors: DesA360P exhibited tensile properties similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited local variations in their tensile properties along the filament length. Based on these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle disease by altering the specific biophysical properties of the desmin filaments, thereby compromising both its mechanosensing and mechanotransduction ability.
The putative transformation of
α-helices into
β-sheets has been studied for more than 50 years in the case of hard
α-keratin. In a previous study of stretched keratin fibers, we specified the ...conditions for
β-sheet appearance within horsehair: the formation of
β-sheets requires at least 30% relative humidity. However, this phenomenon was observed in the whole tissue. Then there was no clear chemical identification of the
β-sheets (keratin or matrix proteins) and the exact location of the
β-sheets across the fiber could not be specified. In this study, using wide-angle x-ray scattering and high spatial resolution infrared microspectroscopy, we could determine and characterize the structural elements across hair sections stretched in water, which provides new information about the aforementioned transition. Our results show that the process can be split into three steps: 1), unraveling of the
α-helical coiled-coil domains, which starts at roughly 5% macroscopic strain; 2), further transformation of the unraveled coiled-coils into
β-sheet structures, which occurs above roughly 20% macroscopic strain; and 3), spatial expanding of the
β-structured zones from the sample center to its periphery.
Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical ...stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.
Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules ...and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a ∼45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (∼3–10 μm) and microtubules (∼1–8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing.
Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 μm and 1 μm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be ∼1 μm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support.
Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.
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•The effect of Al complexation on the solution structures of serum protein is reported here.•Al–protein bindings are via hydrophobic, H-bonding and van der Waals.•More hydrophobic ...b-LG forms more stable complexes than HSA and BSA.•Al cations forced BSA and b-LG into larger or more robust aggregates than HSA.•Al interaction induced larger perturbations of b-LG structure than HSA and BSA.
Al cation is known to induce protein fibrillation and causes several neurodegenerative disorders. We report the spectroscopic, thermodynamic analysis and AFM imaging for the Al cation binding process with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in Al–protein interactions with more hydrophobic b-LG forming stronger Al–protein complexes. Thermodynamic parameters ΔS, ΔH and ΔG showed Al–protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der Waals and H-bonding interactions prevail in HSA and BSA adducts. AFM clearly indicated that aluminum cations are able to force BSA and b-LG into larger or more robust aggregates than HSA, with HSA 4±0.2 (SE, n=801) proteins per aggregate, for BSA 17±2 (SE, n=148), and for b-LG 12±3 (SE, n=151). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced major alterations of protein conformations with the order of perturbations b-LG>BSA>HSA.
•The effect of chitosan nanoparticles on tRNA aggregation and particle formation is studied here.•Chitosan binds tRNA via hydrophilic and electrostatic interactions.•Chitosan-200 forms stronger tRNA ...complexes than chitosan-100 ad chitosan-15kDa.•Major tRNA aggregation and particle formation occurred on chitosan complexation.•tRNA remains in A-family conformation in the presence of chitosan nanoparticles.
The conjugation of tRNA with chitosan nanoparticles of different sizes 15,100 and 200kDa was investigated in aqueous solution using multiple spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed that chitosan binds tRNA via G-C and A-U base pairs as well as backbone PO2 group, through electrostatic, hydrophilic and H-bonding contacts with overall binding constants of KCh-15-tRNA=4.1 (±0.60)×103M−1, KCh-100-tRNA=5.7 (±0.8)×103M−1 and KCh-200-tRNA=1.2 (±0.3)×104M−1. As chitosan size increases more stable polymer-tRNA conjugate is formed. AFM images showed major tRNA aggregation and particle formation occurred as chitosan concentration increased. Even though chitosan induced major biopolymer structural changes, tRNA remains in A-family structure.
Aggregation of trypsin and trypsin inhibitor by Al cation Chanphai, P.; Kreplak, L.; Tajmir-Riahi, H.A.
Journal of photochemistry and photobiology. B, Biology,
April 2017, 2017-Apr, 2017-04-00, 20170401, Letnik:
169
Journal Article
Recenzirano
Al cation may trigger protein structural changes such as aggregation and fibrillation, causing neurodegenerative diseases. We report the effect of Al cation on the solution structures of trypsin ...(try) and trypsin inhibitor (tryi), using thermodynamic analysis, UV–Visible, Fourier transform infrared (FTIR) spectroscopic methods and atomic force microscopy (AFM). Thermodynamic parameters showed Al-protein bindings occur via H-bonding and van der Waals contacts for trypsin and trypsin inhibitor. AFM showed that Al cations are able to force trypsin into larger or more robust aggregates than trypsin inhibitor, with trypsin 5±1 SE (n=52) proteins per aggregate and for trypsin inhibitor 8.3±0.7 SE (n=118). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced more alterations of trypsin inhibitor conformation than trypsin.
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•The aggregation of trypsin and trypsin inhibitor by Al cations was reported here.•Al-protein bindings are via hydrophobic, H-bonding and van der Waals.•More hydrophobic trypsin inhibitor forms more stable aggregate than trypsin.•Al cations forced trypsin and trypsin inhibitor into large and robust aggregates.•Al interaction induced larger perturbations of trypsin inhibitor than trypsin
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