Biophysical Characterization of Tryptophan Locales, Mg²+ Binding and Protein Folding in Gα Subunits

<p>The objective of this study is to understand the structure of guanine nucleotide - binding (G) proteins using a variety of spectroscopic tools. G proteins are membrane-bound proteins consisting of &#945;, &#946;, and &#947; subunits required for the transduction of extracellular signals to various intracellular effectors. Activation of G protein coupled receptors by neurotransmitters or hormones result in a conformational change of a G protein that is triggered by the exchange of guanosine 5'- diphosphate (GDP) bound to the &#61537; subunit for guanosine 5'- triphosphate (GTP) and concomitant dissociation of the &#61538;&#61543; dimer.</p><p>Wild type (WT) Gi&#945;1 has three tryptophan (W) residues. Intrinsic tryptophan fluorescence of WT Gi&#945;1 and the W131F, W211F, and W258F mutants was used to investigate the localized movements of the tryptophan residues. The specific interaction between arginine 208 and W211 that occurred upon activation was measured from the red shift in the emission spectra of each protein. Lastly, tryptic digestion studied conformational protection from proteolysis in all Gi&#945;1 proteins. The results indicated that, upon activation, major fluorescence contribution was from the movement of W211 into a more hydrophobic region, but W131 and W258 were not negligible. In addition, disruption of the W211-Arg208 bridge resulted in complete tryptic digestion.</p><p>We examined the folding mechanism of Gi&#945;1 and Gs&#945; in their inactive and active conformations through various spectroscopic techniques, such as fluorescence, circular dichroism, and UV-Vis spectroscopy. Each respective technique revealed structural insight through changes in the local and global environments of the G&#945; subunits. Overall, G&#945; proteins were more stable in their active conformations and the presence of the electrostatic interaction between W211 and R208 was essential to maintain structural integrity.</p><p>It is known that Mg2+ is required for the formation of the AMF complex, exchange of GTP and GTP hydrolysis in G&#945; proteins. Exploration of Mg2+ dependence on the activation of Gi&#945;1 and Gs&#945; indicated that activation of the AMF complex in G&#945; subunits was more dependent on the presence of Mg2+ than the exchange of GTP&#947;S with GDP.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2013

Author dc:creator

• Najor, Matthew

• Fluorescence
• Folding
• G Proteins
• Lithium
• Magnesium
• Tryptophan
• Biochemistry

Repository record dc:identifier

https://ecommons.luc.edu/luc_diss/730

OAI identifier oai:identifier

oai:ecommons.luc.edu:luc_diss-1729