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Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH


Hofmann, Hagen; Nettels, Daniel; Schuler, Benjamin (2013). Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH. Journal of Chemical Physics, 139(12):121930.

Abstract

The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from -3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect.

Abstract

The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from -3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2013
Deposited On:14 Feb 2014 12:01
Last Modified:23 May 2016 07:22
Publisher:American Institute of Physics
ISSN:0021-9606
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1063/1.4820490
PubMed ID:24089742

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