Circular dichroism, thermal denaturation, and deoxyribonuclease I digestion studies of nucleosomes highly enriched in high mobility group proteins HMG 1 and HMG 2

Biochemistry ◽  
1981 ◽  
Vol 20 (4) ◽  
pp. 1042-1046 ◽  
Author(s):  
James B. Jackson ◽  
Randolph L. Rill
Keyword(s):  
Circular Dichroism ◽  
Thermal Denaturation ◽  
High Mobility ◽  
High Mobility Group ◽  
High Mobility Group Proteins ◽  
Deoxyribonuclease I ◽  
Circular Dichroïsm

scholarly journals Structural organization of DNA–protein complexes of chromatin studied by vibrational and electronic circular dichroism

2010 ◽  
Vol 24 (3-4) ◽  
pp. 239-244 ◽  
Author(s):  
Alexander Polyanichko ◽  
Helmut Wieser
Keyword(s):  
Circular Dichroism ◽  
Protein Complexes ◽  
High Mobility ◽  
Histone H1 ◽  
Cd Spectroscopy ◽  
Linker Histone ◽  
High Mobility Group ◽  
Ir Absorption ◽  
Linker Histone H1 ◽  
Circular Dichroïsm

Structure and functioning of chromatin is determined by interactions of DNA with numerous nuclear proteins. The most abundant and yet not completely understood non-histone chromosomal proteins are those belonging to a High Mobility Group (HMG) namely HMGB1. The interplay of this protein on DNA with linker histone H1 and other proteins determines both structure and functioning of the chromatin. A combination of UV and IR absorption and circular dichroism (CD) spectroscopy was applied to investigate the structure and formation of large supramolecular DNA–protein complexes. This combination of techniques was used to overcome limitations of UV-CD (ECD) spectroscopy due to considerable light scattering in such solutions. Based on the analysis of FTIR and UV circular dichroism spectra and AFM imaging the interaction of DNA with high-mobility group non-histone chromatin protein HMGB1 and linker histone H1 was studied.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

scholarly journals Positioning of nucleosomes containing γ-H2AX precedes active DNA demethylation and transcription initiation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Stephanie Dobersch ◽  
Karla Rubio ◽  
Indrabahadur Singh ◽  
Stefan Günther ◽  
Johannes Graumann ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
High Mobility ◽  
Dna Demethylation ◽  
High Mobility Group ◽  
Regulation Of Transcription ◽  
Dna Breaks ◽  
Transcriptional Initiation ◽  
High Mobility Group Proteins ◽  
Active Dna Demethylation ◽  
Repair Mechanisms

AbstractIn addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.

scholarly journals Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry

2008 ◽  
Vol 9 (1) ◽  
pp. 170-179 ◽  
Author(s):  
Donna R Louie ◽  
Kristen K. Gloor ◽  
Scott C. Galasinski ◽  
Katheryn A. Resing ◽  
Natalie G. Ahn
Keyword(s):  
Mass Spectrometry ◽  
High Mobility ◽  
High Mobility Group ◽  
High Mobility Group Proteins

High mobility group proteins: abundance, turnover and relationship to transcriptionally active chromatin

Biochemistry ◽  
1983 ◽  
Vol 22 (21) ◽  
pp. 5008-5015 ◽  
Author(s):  
Ronald L. Seale ◽  
Anthony T. Annunziato ◽  
Richard D. Smith
Keyword(s):  
High Mobility ◽  
High Mobility Group ◽  
Active Chromatin ◽  
High Mobility Group Proteins
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