Abstract
Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔGU) and the free energy of exchange (ΔGHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔGU and ΔGHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔGU, ΔGU * and residue-specific ΔGHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.html
Highlights
Each protein adopts a specific well-defined three-dimensional (3D) structure, which is important for its biological activities
Where ka, kb and kw are rate constants of acid, base and water catalyzed exchange reactions, respectively; Ra, Rb and Rw are the effect of residues that are on the left and the right sides of amide protons under considerations at acidic, basic and neutral conditions, respectively; pKD is the molar ionization constant of D2O; pD is the pH-meter reading corrected to deuterium effect
Any two cysteins in a protein are considered as cystine, when the distance between the two sulphur atoms of the cysteine residues is within 2.3 A [22] and this particular function of the program has been validated by predicting the cysteine and cystine residues in cardiotoxin III (PDB ID: 2CRT) and cytochrome C (PDB ID: 1HRC, Table 1)
Summary
Each protein adopts a specific well-defined three-dimensional (3D) structure, which is important for its biological activities. The conformational stabilities indicate the free energy differences between the folded (N) and the unfolded conformations (U) of proteins. The stabilization free energies of protein molecules have been determined from the studies of protein unfolding caused by denaturants and temperature [2]. Under a reversible and two-state unfolding process, the population of ‘N’ and ‘U’ of a protein could be precisely estimated by using optical techniques such as fluorescence spectrometry and circular dichroism. The free energy of unfolding (DGU) of proteins is calculated by fitting their unfolded population (U) plotted with respect to denaturant concentration or temperature, to an appropriate two-state model equation [3]. The classical melting analyses provide clues on understanding the mechanism of unfolding (two-state/multi-state processes) and the 3D structural architectures (domains organization) of proteins [4].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
