Heat‐induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first‐order kinetics

Weijers, Mireille; Barneveld, P.A.; Stuart, Martien A. Cohen; Visschers, Ronald W.

doi:10.1110/ps.03242803

Cited by 126 publications

(115 citation statements)

References 37 publications

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“…But simple firstorder aggregation kinetics has been observed previously. (28)(29)(30) Electron microscopy revealed an initial formation of small particles that increased in size to form an amorphous aggregate over the same time frame as the spectral changes (Fig. S4), rather than well-formed fibrils.…”

Section: Discussion Complex Kinetics Fits To a Simple Equation Other mentioning

confidence: 88%

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Wilcken

Wang

Boeckler

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

111

137

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Aggregation of destabilized mutants of the tumor suppressor p53 is a major route for its loss of activity. In order to assay drugs that inhibit aggregation of p53, we established the basic kinetics of aggregation of its core domain, using the mutant Y220C that has a mutation-induced, druggable cavity. Aggregation monitored by light scattering followed lag kinetics. Electron microscopy revealed the formation of small aggregates that subsequently grew to larger amorphous aggregates. The kinetics of aggregation produced surprising results: progress curves followed either by the binding of Thioflavin T or the fluorescence of the protein at 340 nm fitted well to simple two-step sequential first-order lag kinetics with rate constants k 1 and k 2 that were independent of protein concentration, and not to classical nucleation-growth. We suggest a mechanism of first-order formation of an aggregation competent state as being rate determining followed by rapid polymerization with the higher order kinetics. By measuring the inhibition kinetics of k 1 and k 2 , we resolved that the process with the higher rate constant followed that of the lower. Further, there was only partial inhibition of k 1 and k 2 , which showed two parallel pathways of aggregation, one via a state that requires unfolding of the protein and the other of partial unfolding with the ligand still bound. Inhibition kinetics of ligands provides a useful tool for probing an aggregation mechanism.amyloid | misfolding | folding | cancer T he most frequently mutated gene in cancer is that of the tumor suppressor p53. Some 70% of types of human cancers have their p53 directly inactivated by mutation, and so its reactivation is an important therapeutic goal (1). The most common oncogenic mutations are of residues in the DNA binding domain (DBD, residues 94-312) that make direct contact with DNA (2). But about 30%-40% of oncogenic mutants are inactivated because the mutations lower the stability of the DBD so that it denatures at body temperature, although it can be fully active at lower temperatures (3-6). We are attempting to rescue those temperature-sensitive mutants of p53 by designing small molecules that bind specifically to the native state of the protein and stabilize it (7). The rationale is that small molecules that bind to the native state of a temperature-sensitive mutant and not the denatured states will raise the melting temperature by a mass action effect. Such a molecule will slow down the rate of unfolding if it binds weakly to the transition state for unfolding. The obvious binding sites to target are those already present that bind natural ligands. In theory, they can be targeted in a chaperone strategy in which a rescue drug will compete for a binding site (7). We have chosen, instead, as a particularly useful paradigm for these studies, the stability of the p53 mutant Y220C, because the mutation induces a druggable cavity that is remote from functional areas of the protein (8, 9). We have designed small molecules that raise the apparent T m of ...

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Section: Discussion Complex Kinetics Fits To a Simple Equation Other mentioning

confidence: 88%

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Wilcken

Wang

Boeckler

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

111

137

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“…The hB sequence of OVA contains more than 80% hydrophobic amino acids, which is higher than other serpins and may account for its high ␤-aggregation propensity. The hB sequence of OVA is conserved in other members of the serpin superfamily, but the amino acids corresponding to Tyr 42 of OVA are not conserved. The significantly lower ␤-aggregation propensities of human neoroserpin and antithrombin III may be explained by the charged residues present in the region flanking the hydrophobic sequence.…”

Section: Discussionmentioning

confidence: 99%

“…GPC Assay of the Concentration of Non-denatured OVAThe decrease in the concentration of non-denatured OVA under heat-denaturing conditions was determined by GPC assays as reported previously (42). A 0.2 mg/ml OVA solution was incubated at 80°C for various periods of time and then cooled down to room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…The progress of heat-induced aggregation was also monitored by the concentration change in non-denatured OVA solution as estimated from the GPC chromatogram (42). Initially, the large aggregates of OVA were separated from nondenatured protein and small aggregates by centrifugation of the heat-treated OVA solution.…”

Section: Effects Of Disulfide Reduction On Heat-induced Fibrilmentioning

confidence: 99%

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The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

Tanaka

Morimoto

Noguchi

et al. 2011

Journal of Biological Chemistry

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Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys 73 and Cys 120 in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4 -5B are highly ␤-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to ␤-strands, thereby forming sequential intermolecular linkages.The aggregation of misfolded proteins is of significant importance in biology because this phenomenon is closely related to numerous human diseases, including neurodegenerative diseases, such as Alzheimer and Parkinson diseases (1, 2). Amyloid fibril formation of denatured proteins and polymerization of the serpin family of serine protease inhibitors provide well defined structural models of neurodegenerative diseases. The serpins possess a metastable conformation that can undergo a unique conformational change involving the insertion of a proteolytically cleaved reactive center loop into the central ␤-sheets (3, 4), which is required for their inhibitory function. Mutations of serpins lead to their polymerization and intracellular aggregation. Polymer formation of serpins, such as ␣ 1 -antitrypsin, is associated with liver cirrhosis (5), whereas that of neuroserpin (6) results in a cumulative loss of neurons and the onset of Alzheimer-like dementia.The mechanism of serpin polymerization is currently being investigated (7-10). The best described case is provided by the polymerization of the Z variant of ␣ 1 -antitrypsin (11); the Z mutation has a minimal effect on the activity or stability of the native protein (10), and the defect in secretion is due to a perturbation of the folding pathway. A recent structural study of antithrombi...

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“…The application of heat is a wellestablished method that allows protein aggregation to occur on a biochemically feasible time scale. In fact, the thermally induced unfolding of many proteins has been shown to be occasionally followed by an irreversible process that induces aggregation (Sanchez-Ruiz et al 1988;Azuaga et al 2002;Rezaei et al 2002;Weijers et al 2003;Michnik et al 2005). Generally, such aggregation has been modeled as shown in Scheme 1, where N, U, and A are native, unfolded, and irreversible unfolded protein forms, respectively (Lumry and Eyring 1954;Sanchez-Ruiz et al 1988;Azuaga et al 2002;Rezaei et al 2002;Weijers et al 2003;Michnik et al 2005).…”

Section: Thermally Induced Aggregation Of Proteinsmentioning

confidence: 99%

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation

Sasahara

Goto

2012

Biophys Rev

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The aggregation of proteins into amyloid fibrils is a topic that has attracted great interest because the process is associated with the pathology of numerous human diseases. Despite considerable progress in the elucidation of the structure of amyloid fibrils and the kinetic mechanism of their formation, knowledge on the thermodynamic aspects underlying the formation and stability of amyloid fibrils is limited. In this review, we summarize recent calorimetric studies of amyloid fibril formation, with the goal of obtaining a better understanding of the causal factors that thermally induce proteins to aggregate into amyloid fibrils. Calorimetric data show that differential scanning calorimetry is a useful technique to study the causative factors that thermally trigger the conversion to the amyloid structure and highlight the physics related to the thermal fluctuation of proteins during this conversion.

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Heat‐induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first‐order kinetics

Cited by 126 publications

References 37 publications

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation

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