scholarly journals Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data

2005 ◽  
Vol 14 (2) ◽  
pp. 368-374 ◽  
Author(s):  
A. J. Miles
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Spectroscopic Data ◽  
Circular Dichroïsm

scholarly journals Development of advanced chemometric methods for analysis of deep-ultraviolet resonance Raman and circular dichroism spectroscopic data for protein secondary structure determination

2015 ◽  
Author(s):  
◽  
Olayinka Oshokoya
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Least Squares ◽  
Structure Determination ◽  
Spectroscopic Data ◽  
Resonance Raman ◽  
Protein Secondary Structure ◽  
Secondary Structure Content ◽  
Ultraviolet Resonance Raman ◽  
Circular Dichroïsm

Determination of protein secondary structure has become an area of great importance in biochemistry and biophysics as protein secondary structure is directly related to protein function and protein related diseases. While NMR and x-ray crystallography can predict placement of each atom in proteins to within an angstrom, optical methods are the preferred techniques for rapid evaluation of protein secondary structure content. Such techniques require calibration data to predict unknown protein secondary structure content where accuracy may be improved with the application of multivariate analysis. We compare protein secondary structure predictions obtained from multivariate analysis of ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopic data using classical and partial least squares, and multivariate curve resolution-alternating least squares is made. Based on this analysis, the suggested best approach to rapid and accurate secondary structure determination is a combination of both CD and UVRR spectroscopy. While initial studies suggest that a complementary use of spectroscopic data from optical methods such as circular dichroism (CD), infrared (IR) and ultraviolet resonance Raman (UVRR) coupled with multivariate calibration techniques like multivariate curve resolution-alternating least squares (MCR-ALS) is the preferred route for real-time and accurate evaluation of protein secondary structure, further study presents a new strategy for the improvement of secondary structure determination of proteins by fusing CD and UVRR spectroscopic data. Also, a new method for determining the structural composition of each protein is employed, which is based on the relative abundance of the (phi,psi) dihedral angles of the peptide backbone as they correspond to each type of secondary structure. Comparison of the predicted protein secondary structures from MCR-ALS analysis of CD, UVRR and fused data with definitions obtained from dihedral angles of the peptide backbone, yields lower overall root mean squared errors of calibration for helical, sheet, poly-proline II type and total unfolded secondary structures with fused data. Considering that a disadvantage of multivariate calibration methods is the requirement of known concentration or spectral profiles, and second-order calibration methods, such as parallel factor analysis (PARAFAC), do not have such a requirement due to the "second-order advantage", PARAFAC was employed for analysis of UVRR data. An exceptional feature of UVRR spectroscopy is that UVRR spectra are also dependent on excitation wavelength as they are on secondary structure composition. Thus, higher order data can be created by combining protein UVRR spectra of several proteins collected at multiple excitation wavelengths. PARAFAC has been used to analyze UVRR data collected at multiple excitation wavelengths on several proteins to determine secondary structure content.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

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 Investigation by Synchrotron Radiation Circular Dichroism of the Secondary Structure Evolution of Pepsin under Oxidative Environment

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 998
Author(s):  
Laetitia Théron ◽  
Aline Bonifacie ◽  
Jérémy Delabre ◽  
Thierry Sayd ◽  
Laurent Aubry ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Secondary Structure ◽  
Proteolytic Activity ◽  
Digestive Enzyme ◽  
Structure Evolution ◽  
Food Protein ◽  
Maldi Tof ◽  
Kinetics Of ◽  
Circular Dichroïsm

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.

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