Stabilization of protein by replacement of a fluctuating loop: structural analysis of a chimera of bovine alpha-lactalbumin and equine lysozyme.
Abstract: Equine lysozyme is a calcium-binding lysozyme and an evolutional intermediate between non-calcium binding c-type lysozyme and alpha-lactalbumin. We constructed a chimeric protein by substituting the fluctuating loop of bovine alpha-lactalbumin with the D-helix of equine lysozyme. The substitution affects the protection factors not only in the fluctuating loop but also in the antiparallel beta-sheet, the A- and B-helices, and the loop between the B-helix and the beta-sheet. Amide protons in these regions of the chimera are more protected from exchange than are those of bovine alpha-lactalbumin. We used model-free analysis based on 15N nuclear magnetic resonance relaxation measurements to investigate the dynamics of the main chain of the chimera and showed that the fluctuating loop of the chimera is as rigid as three major helices. When we analyzed the chemical shift deviations and backbone HN-H(alpha) scalar coupling constants, we found that the chimera showed an alpha-helical tendency in residues around the fluctuating loop. Our results suggest that the replacement of a highly fluctuating loop in a protein with a rigid structural element in a homologous one may be useful to stabilize the protein structure.
Publication Date: 2002-11-13 PubMed ID: 12427044DOI: 10.1021/bi020360uGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study explores the stabilization of protein structure by replacing a fluctuating loop within bovine alpha-lactalbumin with a structural element from equine lysozyme. The results suggest this substitution could provide a useful method for protein structure stabilization.
Background
- The research is grounded on the study of equine lysozyme, a specific calcium-binding protein considered an evolutionary intermediary between non-calcium binding proteins like c-type lysozyme and alpha-lactalbumin.
- Bovine alpha-lactalbumin contains a fluctuating loop that may impact the protein’s stability.
Methodology
- The researchers synthesized a chimeric protein by replacing the fluctuating loop in bovine alpha-lactalbumin with the D-helix from equine lysozyme, aiming to explore the substitution’s effect on protein stability.
- They employed a model-free analysis based on 15N nuclear magnetic resonance relaxation measurements to study the dynamics of the main chimera chain.
Results
- The substitution affected the protection factors in multiple regions of the protein, including the fluctuating loop, antiparallel beta-sheet, A- and B-helices, and the loop between the B-helix and the beta-sheet.
- As a result of the replacement, amide protons in these regions of the chimera were more protected from exchange than those in bovine alpha-lactalbumin, indicating enhanced stability.
- The dynamics investigation also showed that the fluctuating loop in the chimera was as rigid as the three major helices, suggesting the D-helix’s contribution to protein stabilization.
- Further analysis of chemical shift deviations and backbone HN-H(alpha) scalar coupling constants showed an alpha-helical tendency in residues around the fluctuating loop.
Conclusion
- The study’s results suggested that replacing a highly fluctuating loop of a protein with a rigid structural element from a homologous protein could be a useful method for stabilizing protein structure.
- This could have significant implications for further protein research and engineering, particularly in scenarios where protein stability is a crucial parameter.
Cite This Article
APA
Tada M, Kobashigawa Y, Mizuguchi M, Miura K, Kouno T, Kumaki Y, Demura M, Nitta K, Kawano K.
(2002).
Stabilization of protein by replacement of a fluctuating loop: structural analysis of a chimera of bovine alpha-lactalbumin and equine lysozyme.
Biochemistry, 41(46), 13807-13813.
https://doi.org/10.1021/bi020360u Publication
Researcher Affiliations
- Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan.
MeSH Terms
- Amino Acid Sequence
- Animals
- Cattle
- Circular Dichroism
- Enzyme Stability
- Fluorescent Dyes / metabolism
- Horses
- Lactalbumin / chemistry
- Lactalbumin / metabolism
- Molecular Sequence Data
- Muramidase / chemistry
- Muramidase / metabolism
- Protein Folding
- Protein Structure, Secondary
- Recombinant Fusion Proteins / chemistry
- Recombinant Fusion Proteins / metabolism
- Spectrometry, Fluorescence
Citations
This article has been cited 1 times.- Knappenberger JA, Kraemer-Pecore CM, Lecomte JT. Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture. Protein Sci 2004 Nov;13(11):2899-908.
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