UVM Theses and Dissertations
Format:
Print
Author:
Menke, Andrew
Dept./Program:
Cell and Molecular Biology Program
Year:
2013
Degree:
M.S.
Abstract:
The cyclic GMP dependent protein kinase (PKG) is a critical signaling molecule that is vital in pathways ranging from smooth muscle relaxation to long term potentiation in the nervous system. Biochemical and structural studies of PKG are necessary to further our understanding of these pathways as well as to develop novel forms of therapeutic intervention.
PKG exists in its biological form as a parallel homodimer composed of two identical polypeptides. Prior to the 2011 crystal structure published by the Dostmann lab (Structure, September ih 2011, 1317-1327), no information was available regarding the interaction between PKG peptides or the overall molecular fold of PKG. The published structure contains both of the cyclic nucleotide binding domains ofPKG and also identified a novel domain (the switch helix). This led to our hypothesis that the switch helix domain provides an inter-chain communication site between the two protomers of PKG.
The presence of the switch helix was shown to increase cyclic nucleotide affinity of regulatory constructs of PKG by 100 fold. The modulated binding site was hypothesized to be the carboxy terminal B-site. This result implicates the switch helix as a domain of functional importance that could modulate PKG enzymatic activity. Additional evidence was discovered supporting dimerization of regulatory constructs of PKG in solution using a solvatochromic tryptophan residue located at the switch helix interaction site.
The sequence of PKG was used to create a synthetic switch helix peptide that served as a pharmacological tool to analyze the switch helix interaction site. The switch helix bound specifically to PKG and caused the enzyme to activate in vitro. This is the first time that PKG has been activated by any molecule that is not an analogue of a cyclic nucleotide.
Based on these data, a new model ofPKG activation was created that emphasizes the switch helix domain as a tether that sequesters the catalytic domain of PKG in the enzyme's inactive state. This development can be incorporated into the previous pseudosubstrate catalytic domain disclosure model ofPKG activation. Additionally, the switch helix peptide can be used in the future as a lead compound to develop a powerful pharmacological modulator of PKG function.
PKG exists in its biological form as a parallel homodimer composed of two identical polypeptides. Prior to the 2011 crystal structure published by the Dostmann lab (Structure, September ih 2011, 1317-1327), no information was available regarding the interaction between PKG peptides or the overall molecular fold of PKG. The published structure contains both of the cyclic nucleotide binding domains ofPKG and also identified a novel domain (the switch helix). This led to our hypothesis that the switch helix domain provides an inter-chain communication site between the two protomers of PKG.
The presence of the switch helix was shown to increase cyclic nucleotide affinity of regulatory constructs of PKG by 100 fold. The modulated binding site was hypothesized to be the carboxy terminal B-site. This result implicates the switch helix as a domain of functional importance that could modulate PKG enzymatic activity. Additional evidence was discovered supporting dimerization of regulatory constructs of PKG in solution using a solvatochromic tryptophan residue located at the switch helix interaction site.
The sequence of PKG was used to create a synthetic switch helix peptide that served as a pharmacological tool to analyze the switch helix interaction site. The switch helix bound specifically to PKG and caused the enzyme to activate in vitro. This is the first time that PKG has been activated by any molecule that is not an analogue of a cyclic nucleotide.
Based on these data, a new model ofPKG activation was created that emphasizes the switch helix domain as a tether that sequesters the catalytic domain of PKG in the enzyme's inactive state. This development can be incorporated into the previous pseudosubstrate catalytic domain disclosure model ofPKG activation. Additionally, the switch helix peptide can be used in the future as a lead compound to develop a powerful pharmacological modulator of PKG function.