A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 1988

No description available.

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1992. / Published dissertation. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 81-91).

Thesis (Ph. D.)--University of Lund, 1992. / Published dissertation. Includes bibliographical references.

Cyclic nucleotides such as cAMP and cGMP serve as intracellular second messengers in diverse signaling pathways that control a wide range of cellular functions. Such pathways are regulated by key cyclic nucleotide receptor proteins including protein kinase A (PKA), the exchange protein directly activated by cAMP (EPAC), the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels, and protein kinase G (PKG), and malfunction of these proteins has been linked to a number of pathologies. While it is known that cyclic nucleotide binding to these proteins leads to structural perturbations that promote their activation, the role played by dynamics in auto-inhibition and cyclic-nucleotide-dependent activation is not fully understood. Therefore, in this thesis we examined dynamics within the cyclic-nucleotide receptor proteins EPAC, HCN and PKG, and found that dynamics are critical for allosteric control of activation and/or autoinhibition of all three proteins. In particular, our findings for EPAC and HCN have highlighted dynamics as a key modulator of the entropic and enthalpic components, respectively, of the free-energy landscape for cAMP-dependent allostery, while our findings for PKG have highlighted dynamics as a key determinant of the cGMP-vs.-cAMP selectivity necessary to minimize cross-talk between signaling pathways. Ultimately, we envision that the methods outlined in this thesis will reveal key differences in the regulatory mechanisms of human cyclic nucleotide receptors that can eventually be exploited in the development of novel therapeutics to selectively target a single receptor, and thus treat physiological conditions/diseases linked to malfunction of the target receptor. / Thesis / Doctor of Philosophy (PhD) / In this thesis, we examined cyclic-nucleotide-responsive proteins that regulate key physiological processes, and whose malfunction has been linked to cardiovascular and neurological disorders. In particular, in three such proteins we examined dynamics, whose role in cyclic-nucleotide-responsive function is not fully understood. We found that cyclic-nucleotide-dependent variations in dynamics play a critical role in the function of these proteins, with the results for each protein highlighting a different role played by dynamics. Ultimately, we envision that the methods outlined in this thesis will reveal key functional differences among human cyclic-nucleotide-responsive proteins that can eventually lead to the development of novel therapeutics to treat certain diseases such as arrhythmias or epilepsy by selectively targeting a single cyclic-nucleotide-responsive protein.