scholarly journals Folding mechanism of canine milk lysozyme studied by circular dichroism and fluorescence spectroscopy

2003 ◽  
Vol 17 (2-3) ◽  
pp. 183-193 ◽  
Author(s):  
Masaharu Nakao ◽  
Munehito Arai ◽  
Takumi Koshiba ◽  
Katsutoshi Nitta ◽  
Kunihiro Kuwajima
Keyword(s):  
Circular Dichroism ◽  
Fluorescence Spectroscopy ◽  
Molten Globule ◽  
Guanidine Hydrochloride ◽  
Folding Intermediates ◽  
Equilibrium Unfolding ◽  
Burst Phase ◽  
The Relationship ◽  
Kinetics Of ◽  
Circular Dichroïsm

We have studied the guanidine hydrochloride‒induced equilibrium unfolding and the kinetics of refolding of canine milk lysozyme by circular dichroism and fluorescence spectroscopy. The thermodynamic analysis of the equilibrium unfolding measured by circular dichroism and fluorescence has shown that unfolding is represented by a three‒state mechanism and that the intermediate state of canine milk lysozyme is remarkably more stable than the intermediates observed in other lysozyme and α-lactalbumin. In the kinetic refolding of this protein, there are at least two kinetic intermediates; a burst=phase intermediate accumulated within the dead time (4 ms) of the measurement and an intermediate that has been observed during the kinetics with a rate constant of 10–20 s–1after the burst phase. This result is apparently in contrast with those previously observed in the kinetic refolding of α‒lactalbumin and equine lysozyme that show only the burst‒phase intermediate. The relationship between the extraordinarily stable equilibrium molten globule and the kinetic folding intermediates will be discussed.

Denaturant Induced Equilibrium Unfolding and Conformational Transitional Studies of Germinated Fenugreek β-Amylase Revealed Molten Globule like State at Low pH

2020 ◽  
Vol 27 (10) ◽  
pp. 1046-1057
Author(s):  
Dinesh Chand Agrawal ◽  
Anjali Yadav ◽  
Mohd. Asim Khan ◽  
Suman Kundu ◽  
Arvind M. Kayastha
Keyword(s):  
Circular Dichroism ◽  
Catalytic Domain ◽  
Molten Globule ◽  
Intrinsic Fluorescence ◽  
Tertiary Structures ◽  
Near Ultraviolet ◽  
Equilibrium Unfolding ◽  
Α Helix ◽  
Far Ultraviolet ◽  
Circular Dichroïsm

Background: β-Amylase (EC 3.2.1.2) is a maltogenic enzyme, which releases β-maltose from the non-reducing end of the substrates. The enzyme plays important roles for the production of vaccine, maltiol and maltose rich syrups. Apart from these applications the enzyme protects cells from abiotic as well as oxidative damage. The enzyme is βwell characterized in βplants and microbes and crystal structures of β-amylases βhave been βobtained from sweet potato, soybean and Bacillus cereus. Objective: Find out correlation between structural and functional stability induced by change in pH, temperature and chaotropes. Methods: Activity, intrinsic fluorescence, extrinsic fluorescence, near- and far- ultraviolet circular dichroism spectroscopic measurements were performed. Results: Peaks about 208 nm and 222 nm obtained by near-ultraviolet circular dichroism correspond to α-helix whereas peak at 215 nm shows presence of β-sheet. At pH 2.0, absence of tertiary structures, exposed of hydrophobic regions and presence of substantial secondary structures, revealed the existence of molten globule like state. Temperature induced denaturation studies showed that the enzyme was stable up to 75 ºC and the process was found to be irreversible in nature. Chaotropes dependent equilibrium unfolding studies revealed that at low concentration of chaotropes, ellipticity and intrinsic fluorescence βintensity were βdecreased βwhereas βenzymatic activity remained unchanged, which revealed fenugreek β-amylase is multi-domains enzyme and catalytic βdomain βis more βstable compare to non-catalytic domain. Moreover, the transition was sigmoidal and non-coincidental. Conclusion: Results indicate the probable existence of intermediate states that might perform significant role in physiological process and biotechnological applications.

scholarly journals Conformation Transition Kinetics of Silk Fibroin in Aqueous Solution Explored Using Circular Dichroism Spectroscopy

2021 ◽  
Vol 6 (8) ◽  
pp. 1735-1740
Author(s):  
Sora Lee ◽  
Soo Hyun Kim ◽  
You‐Young Jo ◽  
Wan‐Taek Ju ◽  
Hyun‐Bok Kim ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Circular Dichroism ◽  
Silk Fibroin ◽  
Circular Dichroism Spectroscopy ◽  
Kinetics Of ◽  
Circular Dichroïsm ◽  
Conformation Transition

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.

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.

Stopped-flow circular dichroism

2000 ◽  
Author(s):  
Alison Rodger ◽  
Michael J. Carey
Keyword(s):  
Circular Dichroism ◽  
Detection Methods ◽  
Side Reactions ◽  
Stopped Flow ◽  
Baseline Drift ◽  
Mixing Ratios ◽  
Flow Mixing ◽  
Ideal Tool ◽  
Kinetics Of ◽  
Circular Dichroïsm

As is apparent from previous chapters (Chapters 6, 8 and 9), understanding the kinetics of chemical and biological processes is extremely important. Questions we often consider, explicitly or implicitly, include: Has something happened ‘instantaneously’ or will it take 20 years? Does changing the conditions or available reagents affect either the end product or the rate of a process? What intermediates are produced during a reaction? Can we characterize any intermediates? Do we need to remove them to prevent side reactions? If some or all of the reactants or products are chiral, then circular dichroism (CD) detection may be the ideal tool for following the kinetics of a reaction, and if the half-life of the reaction is of the order of milliseconds to seconds or even minutes then stopped-flow mixing of the reagents will almost certainly be the appropriate choice of sample handling method. For reactions with half-lives of a few minutes to tens of minutes the reagents can be mixed by hand in a normal cuvette and the signal monitored at an appropriate wavelength. CD is not well suited to kinetics on timescales of hours due to the baseline drift that does occur (see Chapter 4, Section 2.5). Some CD spectropolarimeters have the useful facility of being able to perform a wavelength scan at pre-set intervals as well as monitoring continuously (except during the wavelength scan) at a chosen wavelength, thus facilitating the characterization of any intermediates. In this chapter we shall highlight some of the considerations of the stopped-flow technique that are particularly relevant to CD experiments. Particular problems may be encountered when performing CD (as opposed to other detection methods) stopped-flow experiments. The measured signals are very small (typical CD intensities are 0.1% or less of the absorbance signal), and the noise level observed is particularly sensitive to any inhomogeneities or turbulence in the samples. Also, as one of the main applications of stopped-flow CD is in the study of protein folding and unfolding, samples are often very viscous and/or corrosive, have significant absorbances due to buffers etc., and the experiments often require wide and variable mixing ratios.

Structural Differences of Ovalbumin and S-Ovalbumin Revealed by Denaturing Conditions

1997 ◽  
Vol 52 (9-10) ◽  
pp. 645-653 ◽  
Author(s):  
Corrado Paolinelli ◽  
Mario Barteri ◽  
Federico Boffi ◽  
Francesca Forastieri ◽  
Maria Cristina Gaudiano ◽  
...  
Keyword(s):  
Experimental Data ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Guanidine Hydrochloride ◽  
Stable Form ◽  
X Ray ◽  
Heat Stable ◽  
X Ray Scattering ◽  
Structural Differences ◽  
Circular Dichroïsm

We found, by circular dichroism and Raman spectroscopy measurements, that the secondary structure of the native ovalbumin and of its heat-stable form, called S-ovalbumin, is a probe of the structural differences between the two proteins. Small angle X-ray scattering and circular dichroism measurements performed on the two proteins under denaturing conditions, with different concentrations of guanidine hydrochloride, show the changes of the tertiary and secondary structure and a different pathway in the unfolding process. These experimental data confirm that the conversion of native ovalbumin into S-ovalbumin is irreversible and reveal that the response of the two proteins to the same chemical environment is different

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