Journal of Biological Chemistry
Volume 285, Issue 7, 12 February 2010, Pages 4859-4869
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Protein Structure and Folding
Folding Study of Venus Reveals a Strong Ion Dependence of Its Yellow Fluorescence under Mildly Acidic Conditions2

https://doi.org/10.1074/jbc.M109.000695Get rights and content
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Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. By following hydrogen-deuterium exchange of 15N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the β-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching.

Biophysics
Methods/Circular Dichroism CD
Methods/Fluorescence
Methods/NMR
Protein/Conformation
Protein/Denaturation
Protein/Folding
Protein/Thermodynamics

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*

This work was supported in part by Access to Research Infrastructures Activity in the 6th Framework Program of the EC Contract RII3-026145, EU-NMR.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

1

Both authors contributed equally to this work.

3

Recipient of a Studienstiftung des Deutschen Volkes scholarship and supported by the Erasmus/Sokrates Program.

4

National Health and Medical Research Council C. J. Martin Fellow.

5

Supported by The Leverhulme Trust.