Determining the Conformational Stability of a Protein Using Urea Denaturation Curves

2008 ◽  
pp. 41-55 ◽  
Author(s):  
Kevin L. Shaw ◽  
J. Martin Scholtz ◽  
C. Nick Pace ◽  
R. Gerald Grimsley
Keyword(s):  
Conformational Stability ◽  
Urea Denaturation

scholarly journals Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

2005 ◽  
Vol 390 (2) ◽  
pp. 485-492 ◽  
Author(s):  
Vincenzo De Filippis ◽  
Elisa De Dea ◽  
Filippo Lucatello ◽  
Roberta Frasson
Keyword(s):  
Molecular Recognition ◽  
Fluorescence Spectra ◽  
Conformational Stability ◽  
Limited Proteolysis ◽  
Cation Binding ◽  
Molecular Probe ◽  
Free Form ◽  
Urea Denaturation ◽  
Rigid Conformation ◽  
Conformation Stability

In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by ∼2 kcal/mol (1 cal≡4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the ‘fast’ form. Finally, we have used hirudin fragment 1–47 as a molecular probe of the conformation of thrombin recognition sites in the fast and ‘slow’ form. From the effects of amino acid substitutions on the affinity of fragment 1–47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.

scholarly journals DIFFERENTIAL STABILIZING EFFECTS OF BUFFERS ON STRUCTURAL STABILITY OF BOVINE SERUM ALBUMIN AGAINST UREA DENATURATION

2022 ◽  
Vol 52 (1) ◽  
pp. 7-13
Author(s):  
SAAD TAYYAB ◽  
TUAN NOR NAZIAN TUAN MAT ◽  
ADYANI AZIZAH ABD HALIM
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Sodium Phosphate ◽  
Structural Changes ◽  
Conformational Stability ◽  
Urea Denaturation ◽  
Spectral Signal ◽  
Structural Resistance ◽  
Piperazineethanesulfonic Acid

The conformational stability of bovine serum albumin (BSA) against urea denaturation was investigated in aqueous solutions both in the absence and presence of buffers. Various buffers differing in polar and nonpolar characters such as sodium phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this study. Urea-induced structural changes were analyzed using different probes, i.e., intrinsic fluorescence, ANS fluorescence and UV-difference spectral signal.  Presence of different buffers in the incubation medium offered different degrees of resistance to the protein against urea-induced structural changes compared to those obtained in water (in the absence of buffers). A similar trend of buffer-induced structural resistance was noticed with three different probes. The stabilizing effect of these buffers followed the order: MOPS > HEPES > sodium phosphate > Tris-HCl > water. As found in MOPS and HEPES  buffers, the highest stability of BSA can be attributed to the presence of morpholine and piperazine rings, respectively, in their structures. These groups might have produced a hydrophobic environment around the protein surface, thus stabilizing protein conformation against urea denaturation.

Stabilization of Human Serum Albumin against Urea Denaturation by Diazepam and Ketoprofen

2015 ◽  
Vol 22 (7) ◽  
pp. 611-617 ◽  
Author(s):  
Pralad Manoharan ◽  
Yin Wong ◽  
Saad Tayyab
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Urea Denaturation

Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin

2020 ◽  
Vol 27 ◽  
Author(s):  
Edgar Acedo-Espinoza ◽  
Irlanda Lagarda-Diaz ◽  
Rosina Cabrera ◽  
Ana M. Guzman-Partida ◽  
Amir Maldonado-Arce ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Proteolytic Enzymes ◽  
Conformational Stability ◽  
Structural Features ◽  
Beta Sheets ◽  
Hemagglutinating Activity ◽  
Effect Of Temperature ◽  
Hydrodynamic Diameter ◽  
Bioinformatic Tools ◽  
Structural Levels

Background: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. Objective: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. Methods: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. Results: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25 °C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45 °C and exhibited one transition state with a melting temperature of 76.8 °C. Conclusion: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

Conformational Stability, FMO, NMR, MEP and NBO Analysis of 2,5-Di-methyl-2,5-bis(methylthio)-1,4-dithiane and Dimethoxy compounds by DFT Approach

2021 ◽  
Vol 17 ◽  
Author(s):  
Nasrin Masnabadi
Keyword(s):  
Computational Methods ◽  
Electrostatic Potential ◽  
Conformational Stability ◽  
Nbo Analysis ◽  
Dft Methods ◽  
Geometrical Properties ◽  
Stereoelectronic Effect ◽  
Nmr Parameters ◽  
Electron Transfers ◽  
Stability Energy

Abstract: Conformational behaviors of 2,5-dimethoxy-2,5-dimethyl-1,4-dithiane (compound 1) and 2,5-dimethyl-2,5-bis (methylthio)-1,4-dithiane (compound 2) investigated by computational methods including B3LYP/6-311+G** and M06-2X/6-311+G** levels of theory and NBO analysis. The stereoelectronic effect of axial, axial (ax, ax) and equatorial, equa-torial (eq, eq) conformations were studied using NBO analysis. Using NBO analysis, the values of the stereoelectronic effects were calculated through the energy of stability associated with the electron transfers of compounds 1 and 2. The results showed that the eq, eq conformations of the studied compounds were more stable than their corresponding ax, ax conformations, and LP2X→σS1-C2 and LP2S→σ*C2-X electron transfers play important roles in the conformational be-havior of the studied compounds. The main purpose of the present work was to study the effects of stereoelectronic inter-actions and steric on the conformational superiority of the di-methoxy (compound 1) and di-thiomethyl compounds (com-pound 2). Thus, the values of resonance stability energy, non-diagonal elements, and orbital populations were investigated. Also, active electrophilic and nucleophilic centers were identified using fronting orbitals analysis obtained by DFT methods. The electrostatic potential maps of the title compounds were investigated at the B3LYP/6-311+G* level of theory. All of the NMR parameters and geometrical properties of both compounds were determined in this study.

scholarly journals Metal electronic effects on myoglobin conformational stability.

1980 ◽  
Vol 255 (9) ◽  
pp. 4035-4039
Author(s):  
G. McLendon ◽  
P. Murphy
Keyword(s):  
Conformational Stability ◽  
Electronic Effects
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