Project description:This project consists of two experiments. The first is mapping the binding interface between the isolated m-lip domain of mouse lipin and liposomes. The second experiments is mapping the binding interface between full length mouse lipin and liposomes. Looking at the isolated m-lip domain, we found that residues 470-490 and 500-550 showed decreases in exchange upon liposome binding. The full-length lipin experiment saw decreases in exchnage in these same regions, as well as in the very C-terminus and very N-terminus regions of the protein. An order-disorder experiment was done on full length lipin where the protein was exposed to a short pulse of deuterium and compared to the fully-deuterated protein. In this instance, we established that the majority of the protein is relatively disordered and does not have secondary structure with high stability
Project description:This project consisted of three HDX-MS experiments. First, we compared the dimeric PDK1(SKD-PIF) to monomeric PDK1(SKD) and mapped the differences in deuterium incorporation onto the dimer model. We then compared the deuterium incorporation kinetics for the kinase (PDK1(SKD)) and PH (PDK1(PH) domains of PDK1 with full-length PDK1 (PDK1(FL)) in pairwise experiments.
Project description:Use of HDX-MS to study the allosteric and structural differences between the TRAPPII and TRAPPIII complex in the presence and absence of membrane and rab GTPases
Project description:We used Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to examine the dynamic structural changes caused by oncogenic mutations. We performed HDX-MS comparing the full-length complex against the catalytic core which suggest that addition of pY leads to mixed two state population where the state 1 consists of iSH2 bound p110α and the state 2 consists of p110α where the iSH2 is completely disengaged.Therefore, we propose that the non-kinase domain mutations push the equilibrium towards state 2 leading to enhanced enzyme activity.We also propose that the kinase domain mutations lead to the re-orientation of the c-terminal tail leading to exposure of the activation loop and enhanced membrane binding.
Project description:Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is activated by the phospholipid 19 phosphatidylinositol-3-phosphate (PI3P) downstream of growth factor signaling and 20 by Vps34-mediated PI3P production during autophagy. Upregulation of Sgk3 activity 21 has been linked to a number of human cancers due to its overlapping substrate 22 specificity with Akt. Here, we show that Sgk3 is regulated by a combination of 23 phosphorylation and allosteric activation by PI3P. We demonstrate that Sgk3 is 24 specifically activated by PI3P and that PI3P binding induces large conformational 25 changes in Sgk3. Using Vps34 and liposomes containing phosphatidylinositol, we 26 reconstitute Sgk3 signaling via Vps34-mediated PI3P synthesis in vitro. In addition to 27 defining the mechanism of Sgk3 activation by PI3P, our findings open up potential 28 therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.
Project description:Hydrogen deuterium exchange mass spectrometry of PLIN3 in the presence of three different membrane vesicles to analyze structural changes induced by membrane binding.