Insights into the Mechanism of Aggregation and Fibril Formation from Bovine Serum Albumin

Bhattacharya, Mily; Jain, Neha; Mukhopadhyay, Samrat

doi:10.1021/jp111528c

Cited by 178 publications

(166 citation statements)

References 75 publications

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“…In the case of BSA, we used an acidic pH range, as to be far away from the isoelectric point to observe morph aggregation process. The optimum pH was found to be at 3.02; interestingly, a recent study that focuses on characterization of BSA fibrillation mechanism has reported similar results: in the range of 1.6-10.5, pH 3 was found to be the best value [3].…”

Section: Discussionmentioning

confidence: 57%

Response Surface Methodology for Optimizing the Bovine Serum Albumin Fibrillation

et al. 2012

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Amyloid fibrils are considered as nanostructures that could be formed by ordered self-assembly of the partially-folded states of many different peptides or proteins. In this study, bovine serum albumin was used as a model protein whose ordered aggregation (fibrillation) was optimized. Response surface methodology (RSM) was used in a design that contained a total of 30 experimental trials. The first 24 were organized in a factorial design and from 25 to 30 involved the replications of the central points. Data obtained from RSM were subjected to the analysis of variance (ANOVA) and analyzed using a second order polynomial equation. Subsequent testing of the suggested experimental parameters was done in vitro with Congo red spectrophotometric assay. Protein concentration, pH, temperature and time of incubation were the variables used in this study. Responses were assessed by measuring absorbance in 540 nm (characteristic of amyloid formation) and maximal wavelength. Concomitant effects of variables were assessed in surface plots that each considered two of the variables. Interestingly, the pattern obtained by monitoring absorbance at 540 nm and absorbance in maximal wavelength were identical in most cases. We are reporting the optimum concentration of protein, pH, temperature and time at 5 mg ml⁻¹, 3.02 and 72 °C and 48 h, respectively. Our findings suggest that use of Congo red spectrophotometric test, as a simple and affordable assay could be suggested as a first test for assessing fibril formation of proteins. Absorbance in maximal wavelength is recommended as a significant indication of fibril formation.

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Section: Discussionmentioning

confidence: 57%

Response Surface Methodology for Optimizing the Bovine Serum Albumin Fibrillation

et al. 2012

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“…Aggregation and Amyloid Formation by BSA-It was suggested previously that BSA could form amyloid fibrils at low pH and in the presence of high salt conditions (42,43). To further examine the conditions required for BSA amyloid formation, lyophilized BSA (Sigma) was dissolved in PBS, pH 7.4, with a concentration of 21 mg/ml, and the concentration was determined by UV absorption measurement at 280 nm, considering the molar absorptivity of BSA as 43,824 M Ϫ1 cm Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

Jacob¹,

George²,

Singh

et al. 2016

Journal of Biological Chemistry

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Amyloids are highly ordered, cross-␤-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.

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“…The all-α-helical, 583-residue protein BSA ( Fig. 2A) was used for our initial studies as it has well-characterized intrinsic aggregation pathways (28)(29)(30) and its behavior under shear and extensional flow fields has been investigated previously (11,31). To assess whether our extensional flow device can induce protein aggregation, 500 μL gelfiltered monodisperse BSA (Methods) at a concentration of 1, 2, 5, or 10 mg mL −1 was passed through the capillary 500, 1,000, 1,500, or 2,000 times at a plunger velocity of 8 mm s −1 (equivalent to total exposure times to extensional flow of 9, 18, 27, and 36 ms, respectively, at fixed centerline strain-(11,750 s −1 ) and (C) TEM images of 5 mg mL −1 BSA after 0 (Top) and 2,000 passes (Bottom).…”

Section: Design and Computational Characterization Of Extensional Flowmentioning

confidence: 99%

Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

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

129

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Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β 2 -microglobulin (β 2 m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL −1 . In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.extensional flow | aggregation | unfolding | bioprocessing | antibody P roteins are dynamic and metastable and consequently have conformations that are highly sensitive to the environment (1). Over the last 50 y the effect of changes in temperature, pH, and the concentration of kosmatropic/chaotropic agents on the conformational energy landscape of proteins has become well understood (1). This, in turn, has allowed a link to be established between the partial or full unfolding of proteins and their propensity to aggregate (2). The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16). A shearing flow field (Fig. 1A, Top) is caused by a gradient in velocity perpendicular to the direction of travel and is characterized by the shear rate (s −1 ). This results in a weak rotating motion of a protein alongside translation in the direction of the flow. An extensional flow field (Fig. 1A, Bottom) is generated by a gradient in velocity in the direction of travel and is characterized by the strain rate (s −1 ). A protein in this type of flow would experien...

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Insights into the Mechanism of Aggregation and Fibril Formation from Bovine Serum Albumin

Cited by 178 publications

References 75 publications

Response Surface Methodology for Optimizing the Bovine Serum Albumin Fibrillation

Response Surface Methodology for Optimizing the Bovine Serum Albumin Fibrillation

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

Inducing protein aggregation by extensional flow

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