Abstract
Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a “dry molten globule” structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.
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Abbreviations
- Acrylodan:
-
6-Acryloyl-2-dimethylaminonaphthalene
- ATP:
-
Adenosine triphosphate
- cAMP:
-
3′,5′-Cyclic adenosine monophosphate
- kemptide:
-
Peptide substrate for PKAc with the sequence LRRASLG
- MOPS:
-
3-(N-Morpholino)propanesulfonic acid
- PKAc:
-
3′5′-Cyclic adenosine monophosphate dependent protein kinase catalytic subunit
- TRIS:
-
tris(Hydroxymethyl)aminomethane
- GdnHCl:
-
Guanidine hydrochloride
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Acknowledgments
This work was financially supported by Estonian Ministry of Education and Research Grant IUT14-20. The authors are thankful to Prof Per Jemth at Uppsala University for thorough discussion of the work, and Dr. Meeme Utt at Tartu University for help with FPLC analysis.
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Kivi, R., Järv, J. Different States of Acrylodan-Labeled 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions. Protein J 35, 331–339 (2016). https://doi.org/10.1007/s10930-016-9676-8
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DOI: https://doi.org/10.1007/s10930-016-9676-8