The quantitative comparison of the effects of denaturants on the stability of proteins

1986 ◽  
Vol 64 (10) ◽  
pp. 993-998 ◽  
Author(s):  
James A. Thomson ◽  
Charles C. Bigelow
Keyword(s):  
Circular Dichroism ◽  
Specific Binding ◽  
Ribonuclease A ◽  
Quantitative Comparison ◽  
Difference Spectroscopy ◽  
Guanidinium Chloride ◽  
Binding Capability ◽  
The Stability ◽  
Circular Dichroïsm

A novel quantitative comparison of denaturants involving the complete reversible unfolding of proteins is presented. Ribonuclease A was denatured with guanidinium chloride in the presence of low fixed concentrations of various partial denaturants, with the unfolding process being monitored by circular dichroism and difference spectroscopy. The major advantage of this method is that it allows a direct quantitative comparison of the effects of denaturants on the stability of proteins. The effect on the stability of ribonuclease A was shown to be linearly dependent upon the concentration of denaturant. An investigation of the constitutive ions of salts revealed that their effects were additive only in the case of salts that have no specific binding capability. This method can also be useful in detecting the specific binding of salts.

Circular dichroism and infrared study of β-turn formation in repeat peptides of elastin

1987 ◽  
Vol 52 (5) ◽  
pp. 1356-1361
Author(s):  
S. Abdel Rahman ◽  
M. Elsafty ◽  
A. Hattaba
Keyword(s):  
Circular Dichroism ◽  
Solid State ◽  
Ir Spectra ◽  
Chain Length ◽  
Room Temperature ◽  
Infrared Study ◽  
Feature Characteristic ◽  
C 50 ◽  
The Stability ◽  
Circular Dichroïsm

The conformation of elastin-like peptides Boc-Ala-Pro-Gly-Val-APEGM, Boc-Ala-Pro-Gly-Val-Gly-Val-APEGM, Boc-Ala-Pro-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-APEGM, Boc-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-APEGM were examined in solution using circular dichroism at 30 °C, 50 °C, and 70 °C and in solid state by IR at room temperature. The studies show that the β-turn is a significant conformational feature for peptides under investigation in solution at 30 °C and 50 °C, but at 70 °C the tetra, hexa, and decapeptides show the CD feature characteristic of the β-structure while the dodecapeptide spectra show the presence of β-turn which indicates the stability of the β-turn at this chain length. The IR spectra show that in the solid state at room temperature all investigated peptides assume essentially a β-turn except the tetrapeptide which present evidence of antiparallel β-structure. The β-turn contribution in the IR spectra increases with the increase of the chain length of the peptide.

scholarly journals Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus

1987 ◽  
Vol 42 (3) ◽  
pp. 258-262 ◽  
Author(s):  
S. Siebzehnrübl ◽  
R. Fischer ◽  
H. Scheer
Keyword(s):  
Circular Dichroism ◽  
Binding Sites ◽  
Specific Binding ◽  
Reactive Site ◽  
Spectral Changes ◽  
Mastigocladus Laminosus ◽  
Specific Binding Sites ◽  
Close Proximity ◽  
Circular Dichroïsm ◽  
Unambiguous Assignment

C-phycocyanin from the cyanobacterium, Mastigocladus laminosus, and its subunits have been treated with ρ-chloromercuribenzenesulfonate (PCMS). A single reactive site was found on the 13- subunit, and assigned to the single free cystein-β109. The concomitant spectral changes (absorp­tion, fluorescence, circular dichroism), together with the known close proximity of cys-β109 to chromophore β82, allowed an unambiguous assignment of the three spectrally, biochemically and functionally different chromophores to specific binding sites on the two peptide chains (α84: 616-618, β82: 622-624, β153: 598-600 nm).

Circular dichroism studies of sheep β-lipotropic hormone

1976 ◽  
Vol 54 (11) ◽  
pp. 992-998 ◽  
Author(s):  
Serge St-Pierre ◽  
Claude Gilardeau ◽  
Michel Chrétien
Keyword(s):  
Circular Dichroism ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Conformational Transition ◽  
Acidic Solutions ◽  
Nacl Concentration ◽  
Helical Content ◽  
The Stability ◽  
Far Ultraviolet ◽  
Circular Dichroïsm

The far ultraviolet circular dichroism spectra of sheep β-lipotropic hormone (β-LPH) were recorded under different conditions of pH, temperature, salt concentration, and solvent composition. Results confirm the stability of the hormone in strong basic or acidic solutions; moreover, temperatures up to 50 °C do not seem to affect noticeably the conformation of β-LPH. However, increasing the NaCl concentration or addition of dioxane in the solution brings about a conformational transition of the chain, interpreted as an increase in the helical content. The method of Yang (Chen, Y. H., Yang, J. T. &Martinez, H. M. (1972) Biochemistry 11, 4120–4131) was used to compute the proportion of helical, β, and unordered forms of the hormone chain. The proportions are compared with those obtained from Fasman's predictive method (Chou, P. Y. &Fasman, G. D. (1974) Biochemistry 13, 211–221 and Chou, P. Y. &Fasman, G. D. (1974) Biochemistry 13, 222–245) based on the known amino acid sequence of β-LPH.

scholarly journals Optical properties and denaturation by guanidinium chloride and urea of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of four molecular forms of the enzyme

1977 ◽  
Vol 161 (2) ◽  
pp. 321-331 ◽  
Author(s):  
M Nieto ◽  
J A Ayala
Keyword(s):  
Circular Dichroism ◽  
Adenosine Triphosphatase ◽  
Quaternary Structure ◽  
Molecular Form ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Molecular Forms ◽  
Guanidinium Chloride ◽  
Micrococcus Lysodeikticus ◽  
Circular Dichroïsm

1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).

scholarly journals Bis-Lactam Peptide [i, i+4]-Stapling with α-Methylated Thialysines

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4506
Author(s):  
Bo Wu ◽  
Weiping Zheng
Keyword(s):  
Circular Dichroism ◽  
Ribonuclease A ◽  
Rnase A ◽  
Peptide Sequence ◽  
Stability Assessment ◽  
Model Peptide ◽  
Stapled Peptides ◽  
Stapled Peptide ◽  
Proteolytic Stability ◽  
Circular Dichroïsm

Four bis-lactam [i, i+4]-stapled peptides with d- or l-α-methyl-thialysines were constructed on a model peptide sequence derived from p110α[E545K] and subjected to circular dichroism (CD) and proteolytic stability assessment, alongside the corresponding bis-lactam [i, i+4]-stapled peptide with l-thialysine. The % α-helicity values of these four stapled peptides were found to be largely comparable to each other yet greater than that of the stapled peptide with l-thialysine. An l-α-methyl-thialysine-stapled peptide built on a model peptide sequence derived from ribonuclease A (RNase A) was also found to exhibit a greater % α-helicity than its l-thialysine-stapled counterpart. Moreover, a greater proteolytic stability was demonstrated for the l-α-methyl-thialysine-stapled p110α[E545K] and RNase A peptides than that of their respective l-thialysine-stapled counterparts.

scholarly journals Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein

1978 ◽  
Vol 169 (2) ◽  
pp. 371-380 ◽  
Author(s):  
J A Ayala ◽  
M Nieto
Keyword(s):  
Circular Dichroism ◽  
Adenosine Triphosphatase ◽  
Thermal Denaturation ◽  
Enzymic Activity ◽  
Intrinsic Fluorescence ◽  
Guanidinium Chloride ◽  
Subunit Dissociation ◽  
Micrococcus Lysodeikticus ◽  
Maximum Stability ◽  
Circular Dichroïsm

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.

The thermal denaturation of bovine cardiac G-actin

1977 ◽  
Vol 55 (4) ◽  
pp. 325-331 ◽  
Author(s):  
C. C. Contaxis ◽  
C. C. Bigelow ◽  
C. G. Zarkadas
Keyword(s):  
Circular Dichroism ◽  
Conformational Change ◽  
Thermodynamic Analysis ◽  
Thermal Denaturation ◽  
State Transition ◽  
Thermal Unfolding ◽  
Difference Spectroscopy ◽  
Maximum Stability ◽  
Ph Range ◽  
Circular Dichroïsm

The thermal denaturation of bovine cardiac G-actin has been studied by ultraviolet difference spectroscopy and circular dichroism between pH 7.5 and 10.5. As with proteins previously studied, thermal unfolding is incomplete compared with unfolding by urea or GuHCl. However, the same conformational change is observed over the pH range studied, and the available evidence indicates it is a two-state transition. Thermodynamic analysis of the data shows that ΔH0 and ΔS0 are strongly dependent on the temperature, that ΔCp is 1300 cal deg−1 mol−1, and that G-actin has a temperature of maximum stability near −5 °C.

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