Aggregation of Recombinant Human Interferon Gamma: Kinetics and Structural Transitions

Kendrick, Brent S.; Cleland, Jeffrey L.; Lam, Xanthe M.; Nguyen, Tue H.; Randolph, Theodore W.; Manning, Mark C.; Carpenter, John F.

doi:10.1021/js9801384

Cited by 158 publications

(153 citation statements)

References 38 publications

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“…Previous studies on thermal denaturation of BSA in pure buffer have suggested that the protein forms "soluble" aggregates in which a moderate fraction of the native α-helix is transformed into intermolecular β-sheet [17,19,28,29]. A similar trend has been found for numerous other proteins [30][31][32]. At pH values close to neutral, the aggregation of BSA is reaction controlled whereas it becomes faster and diffusion controlled if pH is lowered towards pI, which is ~5 [17,18].…”

Section: Discussionmentioning

confidence: 59%

Solute effects on the irreversible aggregation of serum albumin

Bagger¹,

Ōgendal²,

Westh³

2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractThermal stress on bovine serum albumin (BSA) promotes protein aggregation through the formation of intermolecular β-sheets. We have used light scattering and chromatography to study effects of (<1M) Na 2 SO 4 , NaSCN, sucrose, sorbitol and urea on the rate of the thermal aggregation. Both salts were strong inhibitors of BSA-aggregation and they reduced both the size and number (concentration) of aggregate particles compared to non-ionic solutes (or pure buffer). Hence, the salts appear to suppress both nucleation-and growth rate. The nonelectrolyte additives reduced the initial aggregation rate (compared to pure buffer), but did not significantly limit the extent of aggregation in samples quenched after 27 min. heat exposure

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Section: Discussionmentioning

confidence: 59%

Solute effects on the irreversible aggregation of serum albumin

Bagger¹,

Ōgendal²,

Westh³

2007

Biophysical Chemistry

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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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“…Other studies have used FTIR and CD to follow formation of intermolecular b-sheet structure during protein gellation~Dong et al , 1998Kendrick et al, 1998!. In the present study, we extend this methodology and carry out our experiments on identical samples to enable direct comparisons between the different biophysical techniques. This procedure allows the extreme sensitivity of fibril formation to the solution conditions, and the results of the irreproducible nature of the process, to be overcome in comparing the results of the different techniques.…”

Section: Discussionmentioning

confidence: 99%

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

et al. 2000

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Fourier transform infrared spectroscopy~FTIR!, circular dichroism~CD!, and electron microscopy~EM! have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like a-helical structure. On heating to 70 8C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from a-helical to b-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ;450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.Keywords: CD; electron microscopy; fibril formation; FTIR; insulin; X-ray diffractionThe amyloid diseases, which include Alzheimer's disease, the spongiform encephalopathies and type II diabetes, are characterized by the abnormal self-assembly and deposition of proteinaceous material into insoluble ordered

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Aggregation of Recombinant Human Interferon Gamma: Kinetics and Structural Transitions

Cited by 158 publications

References 38 publications

Solute effects on the irreversible aggregation of serum albumin

Solute effects on the irreversible aggregation of serum albumin

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

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