Burials of ionizable residues inside hydrophobic core are unfavorable in general due to the high desolvation cost of transferring them from water to lipid bilayer. Nevertheless, these residues can form ion-pairs and play vital roles in cellular functions including proton/electron transfer, catalysis, and receptor activation. Proteins in thermophilic microbes maintain their fold and activity in extreme environments like volcanoes, ocean ridges and hot springs with high temperatures (80–110 °C). Previous studies suggested that thermophilic proteins achieve thermostability via an increased number of salt-bridges between ionizable residues compared to their mesophilic homologs. Thus, studies regarding the ionizable residues and thermostability of thermophilic proteins are essential for the fundamental understanding of protein stability, function, and engineering. While related studies have mostly concerned water-soluble proteins, these topics received less attention in membrane proteins. In my dissertation, I aim to bridge the knowledge gaps using the universally conserved rhomboid protease family as a model. I focused on addressing the following two questions: 1) What is the energetic consequence of burying ionizable residue pairs in the core of membrane protein? 2) What is the origin of the thermostability of membrane proteins in thermophilic organisms? In the first question, I found that bearing ionizable residues inside membrane protein induces destabilization of membrane protein kinetically and thermodynamically. Double mutant cycle analysis suggests paired internal ionizable residues form favorable interaction in micelle and bicelle environments. In the second question, my results demonstrate that the delipidated thermophilic rhomboids lose their stability and are fully inactivated at temperatures below their optimal growth conditions compared to mesophilic rhomboid. These results suggest lipids play critical roles in buried ionizable pairs and thermostability in membrane protein folding.
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