scholarly journals Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta

1983 ◽  
Vol 211 (2) ◽  
pp. 295-302 ◽  
Author(s):  
E Odermatt ◽  
J Risteli ◽  
V van Delden ◽  
R Timpl
Keyword(s):  
Human Placenta ◽  
Triple Helix ◽  
Structural Diversity ◽  
Helical Conformation ◽  
Basic Unit ◽  
Bacterial Collagenase ◽  
Disulphide Bonds ◽  
Triple Helices ◽  
Electron Microscopical ◽  
Polypeptide Chains

Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains.

scholarly journals N-Terminal (1→3)-β-d-Glucan Recognition Proteins from Insects Recognize the Difference in Ultra-Structures of (1→3)-β-d-Glucan

2019 ◽  
Vol 20 (14) ◽  
pp. 3498 ◽  
Author(s):  
Adachi ◽  
Ishii ◽  
Kanno ◽  
Tetsui ◽  
Ishibashi ◽  
...  
Keyword(s):  
Triple Helix ◽  
Solid Phase ◽  
Helical Structure ◽  
Helical Conformation ◽  
Insect Host ◽  
Mealworm Beetle ◽  
Indian Meal ◽  
Recognition Protein ◽  
Human Igg1 ◽  
The Difference

Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

scholarly journals Stability of an RNA•DNA–DNA triple helix depends on base triplet composition and length of the RNA third strand

2019 ◽  
Vol 47 (14) ◽  
pp. 7213-7222 ◽  
Author(s):  
Charlotte N Kunkler ◽  
Jacob P Hulewicz ◽  
Sarah C Hickman ◽  
Matthew C Wang ◽  
Phillip J McCown ◽  
...  
Keyword(s):  
Relative Stability ◽  
Triple Helix ◽  
Multiple Factors ◽  
Dna And Rna ◽  
Helix Formation ◽  
Canonical Base ◽  
Triple Helix Formation ◽  
Triple Helices ◽  
Dna Triple Helix ◽  
The Stability

AbstractRecent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA–DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA–DNA triple helices is not well understood beyond the canonical U•A–T and C•G–C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X–Y (where Z = C, U, A, G and X–Y = A–T, G–C, T–A, C–G), in an RNA•DNA–DNA triple helix was determined. The canonical U•A–T and C•G–C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA–DNA triple helix can tolerate up to two consecutive non-canonical A•G–C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA–DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA–DNA and RNA•RNA–RNA triple helices was distinctly different from those in RNA•DNA–DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.

scholarly journals Non-linearity of the collagen triple helix in solution and implications for collagen function

2017 ◽  
Vol 474 (13) ◽  
pp. 2203-2217 ◽  
Author(s):  
Kenneth T. Walker ◽  
Ruodan Nan ◽  
David W. Wright ◽  
Jayesh Gor ◽  
Anthony C. Bishop ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Linear Structure ◽  
Small Degree ◽  
Scattering Data ◽  
Helical Conformation ◽  
End Effects ◽  
Helical Peptides ◽  
Atomistic Modelling ◽  
Triple Helices ◽  
Dynamics Simulations

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly)n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG)10unblocked, was compared with the blocked (POG)10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4–17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease.

scholarly journals Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices

1988 ◽  
Vol 250 (1) ◽  
pp. 65-70 ◽  
Author(s):  
C C Clark ◽  
C F Richards
Keyword(s):  
Chick Embryo ◽  
Triple Helix ◽  
Iron Chelator ◽  
Prolyl Hydroxylase ◽  
Type Ii ◽  
Native Structure ◽  
Lysyl Hydroxylase ◽  
Free Cells ◽  
Triple Helices ◽  
Matrix Free

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2′-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.

Characterization of the cleavage products of human serum immunoglobulin disulphide bonds. I. S-Sulpho derivatives

1967 ◽  
Vol 20 (6) ◽  
pp. 1243 ◽  
Author(s):  
CJ Brackenridge
Keyword(s):  
Human Serum ◽  
Serum Immunoglobulin ◽  
Amino Terminal ◽  
Disulphide Bonds ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Antibody Interactions ◽  
Dispersing Agents ◽  
Time Of Reaction

An examination of variables, including amount of catalyst, pH, sulphite concentration, specific buffer salts, and time of reaction led to conditions for the quantitative sulphitolysis of human serum immunoglobulin disulphide bonds. The cleavage was carried out in the absence of dispersing agents, using air as oxidant and cupric ions as catalyst. The treated protein was characterized by solubility, exclusion chromatography, ultracentrifugation, and immunoelectrophoresis. Separation into three fractions of different molecular weight was achieved by passage through Sephadex G-200 gel. Efforts to eliminate the proportion of aggregated material, which showed no tendency to establish an equilibrium with the remaining two unaggregated fractions, were largely unsuccessful. It was concluded that non-covalent forces play a significant role in antibody interactions. The isolated fractions were individually characterized by sedimentation, heat precipitation, immunoelectrophoresis, and quantitative analysis of amino-terminal residues. This led to the demonstration of at least two polypeptide chains present in each fraction.

scholarly journals The Efek Enzim Bromelin Buah Nanas (Ananas comosus (L.) Merr) Berbasis Sediaan Gel terhadap Lebar Intertubulus Dentin

2020 ◽  
Vol 7 (3) ◽  
pp. 195
Author(s):  
Retno Dewi Alfiyanti ◽  
Berlian Prihatiningrum ◽  
Roedy Budirahardjo
Keyword(s):  
Triple Helix ◽  
Proteolytic Enzymes ◽  
Ananas Comosus ◽  
Maximum Speed ◽  
Specific Enzyme ◽  
Peptide Bonds ◽  
Specific Enzyme Activity ◽  
Lowry Method ◽  
Caries Removal ◽  
Polypeptide Chains

Caries tissue cleaning can use Chemo-Mechanical Caries Removal (CMCR) based on proteolytic  enzymes that catalyze peptide bonds into simpler compounds. Proteolytic enzymes can be found in mature pineapple bromelain enzymes. The aim of the study is to determine the effect of gel-based bromelain enzyme with concentrations of 8%, 10% and 12% against the width of the intertubulus dentin.  The bromelain enzyme is extracted using the Lowry method. Then purified using 80% ethanol and diluting become to concentrations of 8%, 10% and 12%. Diluted bromelain enzymes were formed in gel preparations based on HPMC and applied to the study sample. The results of this study indicated  a  widening of the intertubulus dentin. This is indicated by the variation of the dentin intertubulus width > 2µm. This widening occurs because the bromelain enzyme can hydrolyze collagen in intertubulus dentin in the absence of alpha-l-antitrypsin. Hydrolysis causes the breaking of hydrogen bonds in the triple helix- shaped tropocollagen to turn into strands of polypeptide chains. The bromelain enzyme with a concentration 10% is more effective than 8% and 12% because it  is  within the  maximum speed limit  of  the enzyme so that the enzyme is saturated by its substrate and there is a difference in specific enzyme activity in each concentration.

scholarly journals Practical Guidance in Genome-Wide RNA:DNA Triple Helix Prediction

2020 ◽  
Vol 21 (3) ◽  
pp. 830 ◽  
Author(s):  
Elena Matveishina ◽  
Ivan Antonov ◽  
Yulia A. Medvedeva
Keyword(s):  
Secondary Structure ◽  
Prediction Accuracy ◽  
Triple Helix ◽  
Noncoding Rnas ◽  
Computational Tools ◽  
Cellular Processes ◽  
Genome Wide ◽  
Specific Manner ◽  
Triple Helices ◽  
Practical Guidance

Long noncoding RNAs (lncRNAs) play a key role in many cellular processes including chromatin regulation. To modify chromatin, lncRNAs often interact with DNA in a sequence-specific manner forming RNA:DNA triple helices. Computational tools for triple helix search do not always provide genome-wide predictions of sufficient quality. Here, we used four human lncRNAs (MEG3, DACOR1, TERC and HOTAIR) and their experimentally determined binding regions for evaluating triplex parameters that provide the highest prediction accuracy. Additionally, we combined triplex prediction with the lncRNA secondary structure and demonstrated that considering only single-stranded fragments of lncRNA can further improve DNA-RNA triplexes prediction.

scholarly journals Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation

1996 ◽  
Vol 317 (1) ◽  
pp. 195-202 ◽  
Author(s):  
Neil J. BULLEID ◽  
Richard WILSON ◽  
Janice F. LEES
Keyword(s):  
Triple Helix ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Type Iii ◽  
Helical Domain ◽  
Helix Formation ◽  
Disulphide Bonds ◽  
Type Iii Procollagen ◽  
Triple Helix Formation ◽  
Triple Helical Domain

Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating via their C-propeptides (C-terminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen ‘mini-gene’ which lacks part of the triple-helical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the C-propeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed suggested that inter-chain disulphide bonds had formed within the C-telopeptide (C-terminal telopeptide). However, when hydroxylation of prolyl and lysyl residues was inhibited no inter-chain disulphide bonds were formed in the C-telopeptide, indicating that hydroxylation is required for the initial nucleation of the triple-helical domain. Mutant chains which lacked the ability to form inter-chain disulphide bonds within the C-propeptide or the C-telopeptide could still assemble to form trimeric triple-helical molecules linked by inter-chain disulphide bonds within the N-propeptide (N-terminal propeptide). These results indicate that inter-chain disulphide bond formation within the C-propeptide or the C-telopeptide is not required for chain association and triple-helix formation.

scholarly journals THE NUMBER OF POLYPEPTIDE CHAINS IN BOVINE PLASMA ALBUMIN

1956 ◽  
Vol 34 (2) ◽  
pp. 160-169 ◽  
Author(s):  
M. E. Reichmann ◽  
J. Ross Colvin
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Sodium Chloride ◽  
Sedimentation Velocity ◽  
Performic Acid ◽  
Sedimentation Equilibrium ◽  
Plasma Albumin ◽  
Disulphide Bonds ◽  
Bovine Plasma ◽  
Polypeptide Chains

The molecular weight of performic acid oxidized bovine plasma albumin, dispersed in 0.08 M borate +0.2 M sodium chloride buffer, pH 7.4, was estimated as 30,000 by light-scattering and sedimentation equilibrium methods, 19,000 by osmotic pressure. Sedimentation velocity analyses and electrophoresis showed that the component polypeptide chains of the material are similar in mass and charge density so the polydispersity must be attributed to labile aggregates. The results indicate that here are at least three and probably four similar polypeptide chains in the molecule of native bovine plasma albumin, held together by disulphide bonds.

scholarly journals Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

2016 ◽  
Vol 113 (49) ◽  
pp. 14013-14018 ◽  
Author(s):  
Jessica A. Brown ◽  
Charles G. Kinzig ◽  
Suzanne J. DeGregorio ◽  
Joan A. Steitz
Keyword(s):  
Protein Binding ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Triple Helix ◽  
Rna Binding ◽  
Hek293t Cell ◽  
Proximity Ligation Assay ◽  
Triple Helices ◽  
Gel Shift ◽  
In Cells

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3′-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U•A-U triples interrupted by a C•G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16–MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA–protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 105 molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.

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