Different States of Acrylodan-Labeled 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions.
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.
Institute of Chemistry, University of Tartu, Tartu, Estonia.|Institute of Technology, University of Tartu, Tartu, Estonia.
Rait
R
Kivi
Institute of Chemistry, University of Tartu, Tartu, Estonia. jaak.jarv@ut.ee.
Jaak
J
Järv
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D002021:Buffers; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009025:Morpholines / Q000737:chemistry*; D011489:Protein Denaturation; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D014508:Urea / Q000737:chemistry*
D002021:Buffers; D005456:Fluorescent Dyes; D009025:Morpholines; C008550:morpholinopropane sulfonic acid; C038518:acrylodan; D014508:Urea; D015081:2-Naphthylamine; D017868:Cyclic AMP-Dependent Protein Kinases; D019791:Guanidine
Acrylodan; Catalytic subunit; Deconvolution of fluorescence spectra; Fluorescence spectroscopy; Guanidine hydrochloride; MOPS; Protein denaturation; Urea; cAMP-dependent protein kinase