Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate and that phosphate signal is passed through PhoU to PhoR. Further, this work characterizes residues important for interaction on PhoU and PhoR and identifies a structural model for interaction. This model points to a potential mechanism for PhoU mediated signaling to PhoR. We tested this model with direct coupling analysis and obtained further confirmation. Further use of these techniques may elucidate more of the interactions necessary for proper phosphate signaling.

Nutrient sensing kinases lie at the heart of cellular health and homeostasis, allowing cells to quickly adapt to changing environments. Target of Rapamycin (TOR) and PAS kinase (PASK, or PASKIN) are two such nutrient kinases, conserved from yeast to man. In yeast, these kinases each have paralogs. The two TOR paralogs in yeast mimic the mammalian TORC1 and TORC2 complexes, except both Tor1 and Tor2 may contribute to TORC1 or TORC2 function. The two PAS kinase paralogs are paired with the TOR paralogs, meaning that both Psk1 and Psk2 regulate TORC1, while Psk2 suppresses a temperature-sensitive allele of Tor2. Herein we review the evolutionary models for these paralogs, their function in yeast and mammalian cells, as well as the overlapping function of PAS kinase and TOR. We also use Rice University’s Direct Coupling Analysis algorithms to analyze co-evolutionary relationships and identify potential interaction sites between PAS kinase and several of its substrates.

Does the active membrane transporter, Na+,K+-ATPase dimerize? If it does, whatis the functional benefit? Does it increase or decrease the turnover rate? Theseare still unanswered questions and current research topics. Previous studies havedemonstrated dimerizations in closely related proteins of the P-type ATPase family.For the Na+, K+-ATPase a first indication of dimerization has been shown viaFluorescence lifetime imaging microscopy (FLIM) or Fluorescence resonance energytransfer - Fluorescence correlation spectroscopy (FRET-FCS) experiments. Theprecise dimer structure, dimerization process, and its ultimate functional effecthowever, remain to be found. This master thesis approaches those questions froma co-evolutionary standpoint. It predicts a possible dimer structure by starting with amultiple sequence alignment, direct coupling analysis, and structural contact filteringalgorithm. This model would strengthen the dimerization model of a decreasedturnover rate due to a competitive behavior of two Na+, K+-ATPases for its energysource ATP. / Dimeriserar den aktiva membrantransporten Na+,K+-ATPas? Om den gör det,vad är den funktionella nyttan? Ökar eller minskar det omsättningshastigheten?Dessa är obesvarade frågor och rådande forskningsämnen. Tidigare studier hardemonstrerat dimeriseringar i nära relaterade proteiner av P-typ ATPas-familjen.För Na+, K+-ATPas har en första indikation av dimerisering visats via ”Flourescencelifetime imaging microscopy (FLIM)” eller ”Flourescence resonance energy transfer- Flourescence correlation spectroscopy (FRET-FCS)”. Den precisa dimerstrukturen,dimeriseringsprocessen och dess slutgiltiga funktionella effekt emellertid, återståratt ses. Detta examensarbete på masternivå närmar sig dessa frågor från ettsamevolutionärt perspektiv. Det förutser en möjlig dimerstruktur genom att utgåfrån en flersekvenslinjering, direkt kopplingsanalys och en strukturell kontaktfiltreringsalgoritm. Denna modell skulle stärka dimeriseringsmodellen av minskadomsättningshastighet till följd av tävlingsbeteende mellan två Na+,K+-ATPaser fördess energikälla ATP.