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Tryptophanyl substitutions in apomyoglobin determine protein aggregation and amyloid-like fibril formation at
Ivana Sirangelo1, Clorinda Malmo, Mariateresa Casillo
1Dipartimento di Biochimica e Biofisica, Seconda Università degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy.
Abstract:
Myoglobin is an alpha-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.
Insights
Mutating tryptophan residues in myoglobin to phenylalanine causes protein misfolding and aggregation into amyloid-like fibrils under physiological conditions. This highlights how specific mutations can lead to protein instability and fibril formation.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Misfolding Diseases
Background:
- Myoglobin is an alpha-helical globular protein with conserved tryptophan residues at positions 7 and 14.
- Mutations affecting protein folding can lead to instability and aggregation.
Purpose of the Study:
- To investigate the structural consequences of replacing tryptophan residues (W7F/W14F) in apomyoglobin.
- To determine the effect of these mutations on protein folding, stability, and aggregation propensity.
Main Methods:
- Congo red and thioflavine T binding assays.
- Birefringence and electron microscopy.
- Circular dichroism spectroscopy during refolding experiments.
Main Results:
- The W7F/W14F apomyoglobin mutant was unstable, misfolded, and unable to bind the prosthetic group.
- The mutant protein showed high susceptibility to aggregation and amyloid-like fibril formation at physiological pH.
- Unlike wild-type myoglobin, the mutant aggregated upon pH increase from 4.0 to 7.0, forming beta-structures.
Conclusions:
- Specific mutations affecting protein folding can induce aggregation and amyloid-like fibril formation under physiological conditions.
- The W7F/W14F mutation in apomyoglobin serves as a model for studying mutation-induced protein aggregation.
- Understanding these mechanisms is crucial for comprehending protein misfolding diseases.