scholarly journals Theoretical study of two-photon circular dichroism on molecular structures simulating aromatic amino acid residues in proteins with secondary structures

RSC Advances ◽  
2014 ◽  
Vol 4 (105) ◽  
pp. 60974-60986 ◽  
Author(s):  
Yuly Vesga ◽  
Carlos Diaz ◽  
Florencio E. Hernandez
Keyword(s):  
Circular Dichroism ◽  
Comparative Analysis ◽  
Theoretical Study ◽  
Secondary Structures ◽  
Molecular Structures ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Two Photon ◽  
Α Helix ◽  
Circular Dichroïsm

Calculation and comparative analysis of the theoretical two-photon circular dichroism (TPCD) spectra of l-His, l-Phe, and l-Tyr simulating residues in proteins with secondary structures (α-helix, β-strand and random coil), down to the far-UV region (FUV).

Study of the Effect of Electric Field on Superoxide Dismutase Activity by Spectroscopy

2011 ◽  
Vol 236-238 ◽  
pp. 2445-2448 ◽  
Author(s):  
Qiang Xu ◽  
Miao Wang ◽  
Zhi Huai Yang
Keyword(s):  
Circular Dichroism ◽  
Electric Field ◽  
Biological Effect ◽  
Random Coil ◽  
Sod Activity ◽  
Electric Treatment ◽  
Helix Structure ◽  
Α Helix ◽  
Circular Dichroïsm ◽  
Β Sheet

After the SOD was treated with different strength of electric field, the interactional mechanism of electric field on SOD activity was studied by circular dichroism. The activity of SOD was enhanced under the treatment by different strength of electric field. Circular dichroism spectra showed that the secondary structure of SOD was greatly changed by electric field, as β-sheet and β-turn contents decreased, while α-helix and random coil contents increased. It was considered that the increase of the α-helix structure near the active center would lead to the inactivation of SOD. The result of this study has important meaning to explain the biological effect of electric treatment seeds.

scholarly journals Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

2010 ◽  
Vol 76 (5) ◽  
pp. 1462-1470 ◽  
Author(s):  
Jes�s Fern�ndez-Lucas ◽  
Carmen Acebal ◽  
Jos� V. Sinisterra ◽  
Miguel Arroyo ◽  
Isabel de la Mata
Keyword(s):  
Circular Dichroism ◽  
Lactobacillus Reuteri ◽  
Equilibrium Analysis ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Differential Scanning Calorimetric ◽  
Calorimetric Studies ◽  
Ph Range ◽  
Α Helix ◽  
Circular Dichroïsm

ABSTRACT A novel type II nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% α-helix, 10% β-strand, 16% β-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (Tm ) of 64�C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40�C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2′-fluorodeoxyribonucleosides was carried out.

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.

Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

1998 ◽  
Vol 76 (5) ◽  
pp. 837-842 ◽  
Author(s):  
Daniel Gebreselassie ◽  
Krishna Rajarathnam ◽  
Larry Fliegel
Keyword(s):  
Circular Dichroism ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Cytoplasmic Domain ◽  
Random Coil ◽  
Glutathione S Transferase ◽  
Carboxyl Terminal ◽  
Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

Citrate chelation as a potential mechanism against aluminum toxicity in cells: the role of calmodulin

1985 ◽  
Vol 63 (11) ◽  
pp. 1167-1175 ◽  
Author(s):  
Charles G. Suhayda ◽  
Alfred Haug
Keyword(s):  
Circular Dichroism ◽  
Conceptual Understanding ◽  
Aluminum Toxicity ◽  
Hydrophobic Surface ◽  
Native Structure ◽  
Calcium Calmodulin Dependent ◽  
Α Helix ◽  
Kinetics Of ◽  
Circular Dichroïsm

At a molar excess of [citrate]/[aluminum], this organic acid can protect calmodulin from aluminum binding if the metal is presented to the protein in stoichiometric micromolar quantities, as judged by fluorescence and circular dichroism spectroscopy. Similar citrate concentrations are also capable of fully restoring calmodulin's hydrophobic surface exposure to that of the native protein when calmodulin was initially damaged by aluminum binding. Fluoride anions are equally effective in restoring calmodulin's native structure as determined by fluorescence spectroscopy. Measurements of the kinetics of citrate-mediated aluminum removal also indicated that the metal ions are completely removed from calmodulin, consistent with results derived from atomic absorption experiments. On the other hand, results from circular dichroism studies indicated that citrate-mediated aluminum removal from calmodulin can only partially restore the α-helix content to that originally present in apocalmodulin or in calcium–calmodulin, dependent upon the absence or presence of calcium ions. The results that chelators like citrate can protect calmodulin from aluminum injury may provide a conceptual understanding of physiological observations regarding aluminum-tolerant plant species which are generally rich in certain organic acids.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

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.

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