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A native disulfide stabilizes non-native helical structures in partially folded states of equine β-lactoglobulin.
Abstract: Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.
Publication Date: 2011-11-16 PubMed ID: 22070087DOI: 10.1021/bi2013239Google 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.
- Journal Article
Summary
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The study explores how Equine β-lactoglobulin (ELG) forms non-native helices during its refolding process and in partially folded states. These non-native structures are stabilized by a native disulfide bond, and the research suggests that they only occur during the refolding of a protein with an intact disulfide.
Research Objective and Methodology
- The study was aimed at understanding the structure and folding processes of a particular protein, Equine β-lactoglobulin (ELG).
- Particularly, the researchers focused on understanding non-native helices (protein structures) that ELG assumes in its refolding process and in partially folded states.
- Previous methods such as circular dichroism (CD), applied with site-directed mutagenesis were employed to identify helical regions in ELG that had undergone acid- and cold-related alteration.
- In the current study, the researchers used both CD and nuclear magnetic resonance spectroscopy to examine the structure of a shorter fragment of ELG, which is CHIBLΔF (residues 97-142).
Findings and Conclusions
- The study found that non-native α-helices are located in two distinct ELG regions, residues 98-107 and 114-135. These regions were found to be connected by a native disulfide bond.
- CD spectra of two separate peptides corresponding to these non-native helical regions showed weak helical signatures, with the sum of their spectra being almost similar to the CD spectrum of disulfide-reduced CHIBLΔF.
- These observations led to infer that the non-native helices in ELG are stabilized by this native disulfide bond. This suggests that the formation of non-native helices may only be possible during the protein refolding process when the disulfide bond is intact.
- This conclusion was further supported by the finding that tear lipocalin, a protein similar to ELG but lacking the disulfide bond, did not form non-native helices during its folding process.
By shedding light on the structure and folding process of this particular protein, the study provides valuable insights into potential strategies to manipulate protein structure and function.
Cite This Article
APA
Yamamoto M, Nakagawa K, Fujiwara K, Shimizu A, Ikeguchi M, Ikeguchi M.
(2011).
A native disulfide stabilizes non-native helical structures in partially folded states of equine β-lactoglobulin.
Biochemistry, 50(49), 10590-10597.
https://doi.org/10.1021/bi2013239 Publication
Researcher Affiliations
- Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
MeSH Terms
- Amino Acid Substitution
- Animals
- Circular Dichroism
- Disulfides / chemistry
- Horses
- Lactoglobulins / chemistry
- Lactoglobulins / genetics
- Lactoglobulins / metabolism
- Magnetic Resonance Spectroscopy
- Peptide Fragments / chemistry
- Proline
- Protein Folding
Citations
This article has been cited 2 times.- Ikeguchi M. Transient non-native helix formation during the folding of β-lactoglobulin. Biomolecules 2014 Feb 13;4(1):202-16.
- Yanagida Y, Yoshida K, Ohtomo M, Fujiwara K, Ikeguchi M. Mechanisms of helix induction by the closed loop. Protein Sci 2025 Jun;34(6):e70171.
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