A Fluorescence Spectroscopic and Molecular Dynamics Study of bis-ANS/Protein Interaction

Bothra, Asim K.; Bhattacharyya, Anusree; Mukhopadhyay, Chaitali; Bhattacharyya, Kankan; Roy, Siddhartha

doi:10.1080/07391102.1998.10508216

Cited by 42 publications

(27 citation statements)

References 19 publications

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“…While ANS is the most popular reagent used, its derivative bis-ANS generally shows better fluorescence characteristics (22). These dyes are known to bind to hydrophobic patches on the protein surface (11) largely by means of their aromatic moieties (23). However, some of these, such as ANS and bis-ANS also carry electrostatic charges, and thus have the potential to interact with charged groups on the protein surface.…”

Section: Resultsmentioning

confidence: 99%

Increase in Surface Hydrophobicity of the Cataract-Associated P23T Mutant of Human γD-Crystallin Is Responsible for Its Dramatically Lower, Retrograde Solubility

et al. 2010

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The cataract-associated Pro23 to Thr (P23T) mutation in human γD-crystallin (HGD) has a variety of phenotypes and is geographically widespread. Therefore there is considerable interest in understanding the molecular basis of cataract formation due to this mutation. We showed earlier [Pande, et al. (2005) Biochemistry 44, 2491-2500] that the probable basis of opacity in this case is the severely compromised, retrograde solubility and aggregation of P23T relative to HGD. The dramatic solubility change occurs even as the structure of the mutant protein remains essentially unchanged in vitro. We proposed that the retrograde solubility and aggregation of P23T were mediated by net hydrophobic, protein-protein interactions. Based on these initial findings for P23T and related mutants, and the subsequent finding that they show atypical phase behavior, we concluded that the protein clusters formed in solutions of the mutant proteins were held together by net hydrophobic, anisotropic interactions. Here we show, using chemical probes, that the surface hydrophobicities of these mutants are inversely related to their solubility. Furthermore, by probing the isolated N-terminal domains of HGD and P23T directly, we find that the increase in surface hydrophobicity of P23T is localized in the N-terminal domain. Modeling studies suggest the presence of sticky patches on the surface of the N-terminal domain that could be engaged in forming protein clusters via hydrophobic protein-protein interactions. This work thus provides direct evidence for the dominant role played by net hydrophobic and anisotropic protein-protein interactions in the aggregation of P23T.

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Section: Resultsmentioning

confidence: 99%

Increase in Surface Hydrophobicity of the Cataract-Associated P23T Mutant of Human γD-Crystallin Is Responsible for Its Dramatically Lower, Retrograde Solubility

et al. 2010

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“…It interacts with proteins mainly through its aromatic rings oriented nearly parallel to one anther but not through ionic forces with the sulfonates, which indicates that the binding of BisANS to proteins is predominantly controlled by hydrophobic interactions. Thus, an enhanced binding of BisANS has usually been associated with the states of a protein [13]. Then, successful use of this probe depends upon the development of binding conditions where to enhance hydrophobic interactions between BisANS and the proteins in a relatively constrained environment.…”

Section: 4mentioning

confidence: 99%

Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

et al. 2008

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A simple and sensitive fluorescent staining method for the detection of proteins in SDS-PAGE, namely IB (improved 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) stain, is described. Non-covalent hydrophobic probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non-specifically. As low as 1 ng of protein band can be detected briefly by 30 min washing followed by 15 min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF.

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“…Hydrophobic interactions and electrostatic interactions have both been discussed as the binding mechanism of ANS to proteins . For Bis‐ANS, hydrophobic interactions are seen as the most dominant . However due to the larger size of Bis‐ANS, fluorescence can be inhibited by steric hindrance.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants

Oshinbolu

Shah

Finka

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUNDA current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis‐ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures.RESULTSThe null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis‐ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%. Higher fluorescence intensity on day 14 with the null cultures, also correlated with loss of viability.CONCLUSIONFluorescent dyes are not a specific indicator of mAb aggregation, but rather an indicator of overall protein aggregation or high molecular weight species. SYPRO Orange was more sensitive at detecting very large molecular weight species and ProteoStat seemed better suited to smaller aggregates. Although the assay cannot be used to measure mAb aggregates in cell culture, it could be used to aid cell line selection in maximising viabilities and minimising the amount of aggregates. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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A Fluorescence Spectroscopic and Molecular Dynamics Study of bis-ANS/Protein Interaction

Cited by 42 publications

References 19 publications

Increase in Surface Hydrophobicity of the Cataract-Associated P23T Mutant of Human γD-Crystallin Is Responsible for Its Dramatically Lower, Retrograde Solubility

Increase in Surface Hydrophobicity of the Cataract-Associated P23T Mutant of Human γD-Crystallin Is Responsible for Its Dramatically Lower, Retrograde Solubility

Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants

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