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Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration.
Abstract: The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.
Publication Date: 2003-07-10 PubMed ID: 12850154DOI: 10.1016/s0022-2836(03)00551-5Google 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
- Research Support
- Non-U.S. Gov't
Summary
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This research aims to understand the transformation of calcium-binding equine lysozyme, a type of protein, into amyloid fibrils under acidic conditions and how pH levels and metal ion concentrations influence the formation of their structures.
Experimental Procedures
In the study, equine lysozyme was incubated in an acidic solution and conditions were tweaked to measure the impact of pH, temperature, and calcium concentration on the formation of protofilaments, tiny protein structures which can evolve into fibrils (long, thin fibers). The objective was to explore how these elements influenced the way the protein structures organized.
Amyloid Protofilaments Formation
- The researchers found that at a pH of 4.5 and a temperature of 57 degrees Celsius, the equine lysozyme formed a loosely structured state known as the molten globule state. In this state, protofilaments were formed with a width of around 2 nm. Without the presence of Ca(2+), these protofilaments existed as ring-shaped structures measuring 40-50 nm in diameter.
- However, in a solution containing 10 mM of calcium chloride (CaCl2), the protofilaments took on straight or curved shapes and could cluster into thicker threads but didn’t form into rings.
Fibril Formation at Different pH Levels
- At a lower pH of 2.0, the researchers found that the protein became more destabilised compared to its state at the pH of 4.5. This destabilisation led to the formation of fibrils, or long, thread-like protein structures, at both 37 degrees Celsius and 57 degrees Celsius.
- Also, at this acidic level, both ring-shaped and linear protofilaments were formed, with repeats of approximately 35 nm.
- The ring structures constituted about 10% of all fibrillar species in these conditions and had a larger diameter of 70-80 nm.
Common Characteristics
- All the structures formed in the experiment, including the fibrils and protofilaments, exhibit binding characteristics with Congo red and thioflavine T, research chemicals often used to identify the presence of amyloid fibrils.
- Fibril formation was also accompanied with some acidic hydrolysis, causing the protein to fragment and produce two specific groups of peptides consisting of residues 1-80 and 54-125.
Conclusion
The resulting fibril structure was more commonly filled with full-length equine lysozyme at the early stages of the experiment. The research suggests that the ring-shaped structures observed could constitute a secondary generic type of amyloid structure along with the more often observed linear amyloid fibrils. This mimicry of pathologic processes can provide a basis for looking at disease development and potential intervention strategies.
Cite This Article
APA
Malisauskas M, Zamotin V, Jass J, Noppe W, Dobson CM, Morozova-Roche LA.
(2003).
Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration.
J Mol Biol, 330(4), 879-890.
https://doi.org/10.1016/s0022-2836(03)00551-5 Publication
Researcher Affiliations
- Department of Biochemistry, Umeå University, Umeå SE-90187, Sweden.
MeSH Terms
- Amyloid / chemistry
- Animals
- Benzothiazoles
- Blotting, Western
- Calcium / metabolism
- Coloring Agents / pharmacology
- Congo Red / pharmacology
- Crystallography, X-Ray
- Electrophoresis, Polyacrylamide Gel
- Horses
- Hydrogen-Ion Concentration
- Hydrolysis
- Ions
- Microscopy, Atomic Force
- Models, Molecular
- Muramidase / chemistry
- Protein Conformation
- Protein Structure, Tertiary
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Temperature
- Thiazoles / chemistry
Citations
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