UVM Theses and Dissertations
Format:
Print
Author:
Raidas, Shiv Kumar
Title:
Dept./Program:
Cell and Molecular Biology Program
Year:
2009
Degree:
M.S.
Abstract:
CGMP-dependent protein kinase (PKG) belongs to the family of basophilic serine threonine kinases and is involved in numerous physiological functions ranging from smooth muscle contraction to platelet aggregation and neuronal signal transduction. PKG shares structural and functional homology to CAMP-dependent protein kinase (PKA) which mediates vasorelaxation effects similar to PKG but through different pathways. To distinguish the PKG and PKA mediated pathways in the control of vasoreactivity, potent and specific inhibitors are required. Our laboratory has developed the substrate competitive peptidic inhibitors, DT-2 and D-DT-2 which are the most potent and specific PKG inhibitors known today [1, this study]. DT-2 contains the high affinity peptide for PKG, LRKKKKKH, (W45) and the internalization sequence from HIV tat, YGRKKRRQRRRPP (DT-6).
In D-DT-2, all amino acids have reversed, Dconfiguration. DT-2 has been utilized in numerous studies to investigate PKG mediated effects in smooth muscle contractility. However, the exact mechanism of inhibition mediated by this blocker has remained obscure. Here, we hypothesize that DT-2 has unique modes of inhibiting PKG. Dixon plot analysis of DT-2 and D-DT-2 show a hitherto unique case of enzymatically active enzyme-substrate-inhibitor [ESI] complex. Moreover, Schild and Eadie Hofstee kinetic analyses revealed that the mechanism of inhibition mediated by DT-2 .is mixed competitive/non-competitive. We found that the competitive and non-competitive elements of inhibition were contributed by the W45 and DT-6 parts respectively. Taken these results together, we developed a hitherto unrecognized hyperbolic mixed inhibition model containing a productive ESI complex for DT-2 mediated PKG inhibition.
Furthermore, we attempted to develop new analogs of DT-2 based on the criteria of charge, length, backbone and internalization sequence. We hypothesized that by altering the charge, backbone or internalization sequence of DT-2, without modifying the high affinity W45 sequence, inhibitors of comparable PKG inhibitory potential could be generated. These DT-2 analogs are potential candidates for PKG blockers with properties of optimized cellular permeability and /or improved proteolytic stability.
Additionally, we investigated the conformation by which peptide substrates bind PKG. We hypothesized that PKG recognizes peptidic substrates with a left-handed polyproline type II (PP II) conformation. Analogs based on the PKG recognition sequences RRXSI and RKKSI [X=any amino acid] with incorporated PP II character [through proline ternplated amino acids] were tested as PKG substrates. Our results show that these analogs do act as PKG substrates. However, they appear to disfavor the PP II character at P-2 position. This finding will potentially have implications in generating future agonists/antagonists of PKG with optimized binding conformation.
Finally, we attempted to study PKG: DT-2 binding by combining the techniques of photo-affinity labeling and mass spectrometry. We synthesized benzophenone photoaffinity labeled DT-2 analogs to study it's interactions with the kinase to explain the unique 1:2 stoichiometry of DT-2: PKG binding reported previously. We developed a protocol for identification of DT-2 binding sites on PKG by utilizing MALDI and LC mass spectrometry after separate digestions of PKG: DT-2 complex by trypsin and chymotrypsin. Moreover, we identified the exact mass additions to search for in labeled peptides by MALDI analysis of trypsin digested DT-2 photolables alone.
In D-DT-2, all amino acids have reversed, Dconfiguration. DT-2 has been utilized in numerous studies to investigate PKG mediated effects in smooth muscle contractility. However, the exact mechanism of inhibition mediated by this blocker has remained obscure. Here, we hypothesize that DT-2 has unique modes of inhibiting PKG. Dixon plot analysis of DT-2 and D-DT-2 show a hitherto unique case of enzymatically active enzyme-substrate-inhibitor [ESI] complex. Moreover, Schild and Eadie Hofstee kinetic analyses revealed that the mechanism of inhibition mediated by DT-2 .is mixed competitive/non-competitive. We found that the competitive and non-competitive elements of inhibition were contributed by the W45 and DT-6 parts respectively. Taken these results together, we developed a hitherto unrecognized hyperbolic mixed inhibition model containing a productive ESI complex for DT-2 mediated PKG inhibition.
Furthermore, we attempted to develop new analogs of DT-2 based on the criteria of charge, length, backbone and internalization sequence. We hypothesized that by altering the charge, backbone or internalization sequence of DT-2, without modifying the high affinity W45 sequence, inhibitors of comparable PKG inhibitory potential could be generated. These DT-2 analogs are potential candidates for PKG blockers with properties of optimized cellular permeability and /or improved proteolytic stability.
Additionally, we investigated the conformation by which peptide substrates bind PKG. We hypothesized that PKG recognizes peptidic substrates with a left-handed polyproline type II (PP II) conformation. Analogs based on the PKG recognition sequences RRXSI and RKKSI [X=any amino acid] with incorporated PP II character [through proline ternplated amino acids] were tested as PKG substrates. Our results show that these analogs do act as PKG substrates. However, they appear to disfavor the PP II character at P-2 position. This finding will potentially have implications in generating future agonists/antagonists of PKG with optimized binding conformation.
Finally, we attempted to study PKG: DT-2 binding by combining the techniques of photo-affinity labeling and mass spectrometry. We synthesized benzophenone photoaffinity labeled DT-2 analogs to study it's interactions with the kinase to explain the unique 1:2 stoichiometry of DT-2: PKG binding reported previously. We developed a protocol for identification of DT-2 binding sites on PKG by utilizing MALDI and LC mass spectrometry after separate digestions of PKG: DT-2 complex by trypsin and chymotrypsin. Moreover, we identified the exact mass additions to search for in labeled peptides by MALDI analysis of trypsin digested DT-2 photolables alone.