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The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme.
Abstract: The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins: hen egg white lysozyme and alpha-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions called the alpha and beta domains. In equine lysozyme and alpha-lactalbumin the latter domain specifically binds Ca2+. It is shown that, in contrast to hen egg white lysozyme in which the alpha and beta domains unfold as a single cooperative unit, in equine lysozyme the two domains unfold in two separate cooperative stages even in the presence of excess Ca2+. The calcium binding beta-domain unfolds at a lower temperature and with more extensive heat absorption than the alpha-domain. Binding of Ca2+ increases the stability of the beta-domain, but even in the holo form it is less stable than the alpha-domain. The thermodynamic characteristics of Ca2+ binding have been determined, and indicate that it is an entropically driven process. The unfolding of equine lysozyme largely resembles the unfolding of alpha-lactalbumin, which also unfolds in two stages, but in the latter case the second stage is much less cooperative and proceeds with a smaller and diffuse heat absorption. As a result, the total enthalpy of unfolding of equine lysozyme is significantly larger than that of alpha-lactalbumin, being almost of the same magnitude as the enthalpy of egg white lysozyme unfolding, which proceeds as a single two-state transition. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca2+, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperativity in equine lysozyme is not the cluster of negative charges forming the calcium binding site, but the difference in atomic packing in the interior and at the interface between the alpha and beta domains.
Publication Date: 1995-09-29 PubMed ID: 7563064DOI: 10.1006/jmbi.1995.0510Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research assesses the unfolding thermodynamics of c-type lysozymes, with a specific focus on equine lysozyme. Using calorimetric methods, it compares the heat induced unfolding of equine lysozyme with structurally similar proteins, hen egg white lysozyme and alpha-lactalbumin. The study uncovers differences in how various domains of these proteins react to temperature change and identifies contributing factors.
Study Approach and Observations
- The researchers conducted a calorimetric study to examine the energetics of temperature-induced unfolding of the protein equine lysozyme.
- They compared these findings to two proteins with a similar molecular structure, hen egg white lysozyme and alpha-lactalbumin.
- All of the proteins in the study have a structure that is characterized by a deep cleft, dividing the molecule into two regions referred to as the alpha and beta domains. In equine lysozyme and alpha-lactalbumin, the beta domain specifically binds with Ca2+.
- They found that, unlike hen egg white lysozyme where the alpha and beta domains unfold together, in equine lysozyme the domains unfold in two separate stages, even when there is an excess of Ca2+. The calcium binding beta-domain unfolds at lower temperatures with more heat absorption than the alpha-domain.
The Role of Calcium and Domain Stability
- It is shown that the binding of Ca2+ increases the stability of the beta-domain, but even in its holo form, the beta-domain is less stable than the alpha-domain.
- The researchers determined the thermodynamic attributes of Ca2+ binding, showing that it is driven by entropy.
Comparison with Alpha-lactalbumin
- They also found that the unfolding process of equine lysozyme strongly resembles that of alpha-lactalbumin, which also unfolds in two stages. However, the second stage in the latter protein is less cooperative and comes with a smaller and more diffuse heat absorption.
- As a result, the full enthalpy of unfolding for equine lysozyme is substantially larger than that of alpha-lactalbumin. Its enthalpy is nearly the same as the enthalpy of egg white lysozyme unfolding, which takes place as a single two-state transition.
Findings and Conclusions
- Reviewing the unfolding enthalpy function of the different lysozymes – those that do or do not bind with Ca2+ and those that unfold in one or two stages – the researchers arrived at the conclusion that the principal reason for the loss of interdomain cooperativity in equine lysozyme isn’t the cluster of negative charges formulating the calcium binding site. Rather, they attribute it to differences in atomic packing in the interior and the interface between the alpha and beta domains.
Cite This Article
APA
Griko YV, Freire E, Privalov G, van Dael H, Privalov PL.
(1995).
The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme.
J Mol Biol, 252(4), 447-459.
https://doi.org/10.1006/jmbi.1995.0510 Publication
Researcher Affiliations
- Department of Biology and Biocalorimetry Center, Johns Hopkins University Baltimore, MD 21218, USA.
MeSH Terms
- Animals
- Binding Sites
- Calcium / metabolism
- Calorimetry
- Chickens
- Horses
- Lactalbumin / chemistry
- Models, Molecular
- Muramidase / chemistry
- Ovum / chemistry
- Protein Denaturation
- Protein Folding
- Protein Structure, Tertiary
- Thermodynamics
Grant Funding
- GM48036-01 / NIGMS NIH HHS
- RR04328 / NCRR NIH HHS
Citations
This article has been cited 14 times.- Levartovsky Y, Shemesh A, Asor R, Raviv U. Effect of Weakly Interacting Cosolutes on Lysozyme Conformations. ACS Omega 2018 Nov 30;3(11):16246-16252.
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- Olalere OA, Guler F, Chuck CJ, Leese HS, Castro-Dominguez B. Mechanochemical extraction of edible proteins from moor grass. RSC Mechanochem 2024 Sep 10;1(4):375-385.
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