Related Experiment Video
Updated: Aug 15, 2026
05:48
Rapid Generation of Amyloid from Native Proteins In vitro
Published on: December 5, 2013
Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution
Charles Chow1, Nese Kurt, Regina M Murphy
1Department of Chemistry, and Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Biophysical Journal
|October 11, 2005
Summary
This study characterizes nonfunctional apomyoglobin (apoMb) aggregates, revealing their alpha-helical or random coil structures. Protein refolding can lead to both native monomers and misfolded, self-associated states.
Area of Science:
- Biophysics
- Protein Chemistry
- Biochemistry
Background:
- Understanding protein folding and misfolding is crucial for disease research.
- Characterizing nonfunctional protein aggregates provides insights into these processes.
- Physiologically relevant conditions are needed for accurate biophysical studies.
Purpose of the Study:
- To biophysically characterize nonfunctional apomyoglobin (apoMb) soluble aggregates at room temperature.
- To investigate the structural and kinetic properties of apoMb aggregates under varying solution conditions.
- To elucidate the refolding pathways of apoMb, including the formation of misfolded states.
Main Methods:
- Dynamic and static laser light scattering were used for aggregate detection and characterization.
- Apomyoglobin (apoMb) solutions were prepared in buffer at pH 6.0, with and without high urea concentrations.
- Structural analysis involved assessing secondary structure (alpha-helical, random coil, beta-sheet) and hydrodynamic diameters.
Main Results:
- Soluble apoMb aggregates with hydrodynamic diameters of 80-130 nm and semiflexible, chain-like morphology were detected at room temperature.
- Unlike previously reported high-temperature aggregates, these soluble aggregates exhibited alpha-helical or random coil secondary structures.
- Low pH and high urea concentration unfolded the protein and eliminated aggregates, but refolding still produced misfolded, self-associated species.
- Kinetic refolding proceeded via parallel routes, yielding native monomers and misfolded, self-associated states with native-like secondary structure.
Conclusions:
- Apomyoglobin (apoMb) can form distinct soluble aggregates at room temperature with varying secondary structures.
- Protein unfolding and refolding dynamics are complex, with a propensity to form misfolded, self-associated states even from an unfolded monomer.
- These findings highlight the importance of solution conditions in protein aggregation and misfolding pathways.
Related Concept Videos
01:20Protein Buffers in Blood Plasma and Cells
The human body utilizes protein buffer systems to maintain a stable pH. These systems capitalize on the dual role of amino acids, which can act as acids or bases by accepting or releasing hydrogen ions in response to pH changes. Protein buffer systems are particularly significant in the extracellular fluid (ECF) and intracellular fluid (ICF) of active cells, where structural and functional proteins provide substantial buffering capacity.
Certain amino acids can exist in a zwitterion state at a...
Certain amino acids can exist in a zwitterion state at a...
01:55What are Proteins?
Overview
02:15Protein and Protein Structure
Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective. They may serve in transport, storage, or membranes; or they may be toxins or enzymes. Their structures, like their functions, vary greatly. They are all, however, amino acid polymers arranged in a linear sequence.
A protein's shape is critical to its function. For example, an enzyme can...
A protein's shape is critical to its function. For example, an enzyme can...
01:28Protein Denaturation
The function of proteins depends on their native three-dimensional structure, which is dictated by the amino acid sequence of the specific protein. Folding of the polypeptide chain takes place under specific conditions that energetically favor the folded conformation. In contrast, protein denaturation occurs spontaneously under unfavorable conditions that disrupt the integrity of the folded conformation. Thus, the chemical and physical environment of a protein, such as significant changes in pH...
01:27SDS-PAGE
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
01:25Basicity of Heterocyclic Aromatic Amines
Heterocyclic amines, where the N atom is a part of an alicyclic system, are similar in basicity to alkylamines. Interestingly, the heterocyclic amine having a nitrogen atom as part of an aromatic ring has much less basicity than its corresponding alicyclic counterpart. For this reason, as presented in Figure 1, piperidine (pKb = 2.8) is significantly more basic than pyridine (pKb = 8.8).