Kinetics of Acrylodan-Labelled cAMP-Dependent Protein Kinase Catalytic Subunit Denaturation

Kivi, Rait; Loog, Mart; Jemth, Per; Järv, Jaak

doi:10.1007/s10930-013-9511-4

Cited by 9 publications

(10 citation statements)

References 15 publications

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“…Buffers were made using Milli-Q deionized water. Acrylodan-labeled PKAc was prepared by the labeling of the PKAc N32C mutant with acrylodan (Anaspec, USA, Cat# 83305, USA) as described in detail in our previous paper [7].…”

Section: Chemicalsmentioning

confidence: 99%

“…Enzyme concentration ion all assays was 50 nM. The weak Raman emission of water at around 465 nm was subtracted from the spectra before data processing, as described in [7].…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

“…Kinetic runs were started by addition of the enzyme into the assay buffer containing MOPS and necessary ligands, and the fluorescence of the sample was monitored as long as the degradation process occurred. These experiments were described in more detail way in our previous study [7].…”

Section: Kinetics Of Acrylodan-labeled Pkac Denaturationmentioning

confidence: 99%

“…In the present report we filled this gap and studied interaction of a series of peptide inhibitors with the peptide binding site on the free PKAc by measuring the stabilizing effect of these ligands on the kinetics of enzyme denaturation [7]. This approach employs the principles of chemical denaturation shift, proposed for the screening of enzyme-ligand interactions [8], and was previously used for characterization of binding of peptide inhibitor PKI with PKAc [7].…”

Section: Introductionmentioning

confidence: 99%

“…This approach employs the principles of chemical denaturation shift, proposed for the screening of enzyme-ligand interactions [8], and was previously used for characterization of binding of peptide inhibitor PKI with PKAc [7].…”

Section: Introductionmentioning

confidence: 99%

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Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits

et al. 2016

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The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

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Section: Chemicalsmentioning

confidence: 99%

“…Enzyme concentration ion all assays was 50 nM. The weak Raman emission of water at around 465 nm was subtracted from the spectra before data processing, as described in [7].…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Section: Kinetics Of Acrylodan-labeled Pkac Denaturationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits

et al. 2016

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Thermodynamic Aspects of cAMP Dependent Protein Kinase Catalytic Subunit Allostery

2014

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Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5-24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5-50 °C). The thermodynamic parameters ΔH and ΔS for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van't Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5-24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (ΔH). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy-enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.

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Different States of Acrylodan-Labeled 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions

Kivi

Järv

2016

Protein J

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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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Kinetics of Acrylodan-Labelled cAMP-Dependent Protein Kinase Catalytic Subunit Denaturation

Cited by 9 publications

References 15 publications

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits

Thermodynamic Aspects of cAMP Dependent Protein Kinase Catalytic Subunit Allostery

Different States of Acrylodan-Labeled 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions

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