Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Dynamics of allosteric regulation of the phospholipase C-gamma isoform

ABSTRACT: we used hydrogen deuterium exchange masss spec (HDX-MS) to define the exchange kinetics of PLC-y1 as it interacts with the kinase domain of FGFR1, and liposomes containing PIP2, in order to understand how PLC-y isozymes operate at membranes. We show that the binding of FGFR1k to PLC-y1 increases deuterium incorporation within the interface between the regulatory domains and the catalytic core. Importantly, the addition of liposomes further increase deuterium exchange within the same region.

INSTRUMENT(S): impact HD

ORGANISM(S): Rattus Norvegicus (rat)

SUBMITTER: John Burke

LAB HEAD: Dr John E Burke

PROVIDER: PXD030492 | Pride | 2022-06-14

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	01042019_RHHDX22_PLC_D1165H_CD_MSMS_25pmol_DBSEARCH_7.zip	Other
	01042019_RHHDX22_PLC_WT_and_D1165H__-peptide_-Lipid_-FGFR.hdx	Other
	01042019_RHHDX22_PLC_WT_and_D1165H__-peptide_-Lipid_-FGFR_MutationPeptidesOnly.hdx	Other
	01_4_19_RHHDX22_PLC_A_30s_1_0_01_9572.d.zip	Other
	01_4_19_RHHDX22_PLC_B_30s_1_0_01_9574.d.zip	Other

Items per page:

1 - 5 of 131

Similar Datasets

HDX-MS analysis of PLIN3 membrane binding

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.

2023-04-14 | PXD025717 | Pride

Structural Basis for Defective Membrane Targeting of Mutant Enzyme in Human VLCAD Deficiency

Project description:Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function analysis, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, and computational modeling, to identify a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted a-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, we find that enzyme mislocalization underlies the metabolic deficiency syndrome of patients bearing specific mutations that disrupt the structure of an a-helical membrane binding region of VLCAD.

2022-06-29 | PXD029565 | Pride

Characterization of m-lipin using HDX-MS

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

2021-06-29 | PXD022172 | Pride

Hydrogen Deuterium Exchange of PDK1

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.

2022-05-20 | PXD027401 | Pride

Molecular basis for differential activation of p101 and p84 complexes of PI3Kg by Ras and GPCRs

Project description:Using hydrogen deuterium exchange mass spectrometry, we reveal molecular differences between the two p110g-p84 and p110g-p101 complexes that explain their differential activation.

2023-03-09 | PXD035723 | Pride

HDX-MS Analysis of the protein ALT51

Project description:HDX-MS analysis of the protein ALT51, comparing deuterium uptake between the full length protein and a truncated construct missing the CBM-like domain

2023-02-22 | PXD034850 | Pride

An HDX-MS optimised approach to characterise nanobodies as tools for biochemical and structural studies

Project description:Given the far-reaching applications of nanobodies, a stream-lined approach to characterizing nanobodies tailored to its intended application is warranted. Camelids can produce a large number of antibodies that can bind to a host of epitopes spanning the entire protein of interest, making identification of binders appropriate to your study slow and cumbersome. To overcome this challenge, we propose the use of hydrogen-deuterium mass spectrometry (HDX-MS) to rapidly characterize nanobody epitopes. HDX-MS is a technique that measures the rate of exchange of amide hydrogens in the protein backbone and can provide valuable insight into interaction surfaces with binding partners. Using the example of the lipid signalling complex PI3K, we employed HDX-MS to characterize the epitopes of a large cohort of nanobodies simultaneously with reasonable accuracy. Some of these nanobodies proved to be extremely useful, stabilizing a flexible region in structural studies and blocking signalling inputs from activating partners in in-vitro kinase assays. These experiments were in turn, instrumental in obtaining the first high resolution structure of a PI3K complex, besides providing novel tools to study PI3K signalling in-vivo.

2021-09-22 | PXD025207 | Pride

Hydrogen Deuterium Exchange of PI4K(530-560) Peptide with Calcineurin

Project description:we used HDXMS to study if PI4KA(530-560) is able to bind calcineurin. Here we showed a significant protection in deuterium exchange upon binding calcineurin in the wild type state, indicating these proteins are interacting. Using a mutant that should inhibit the interaction (AKASAA), we showed that this mutant does in fact prevent protection by calcineurin.

2024-09-03 | PXD044636 | Pride

Detailing the Structural and Dynamic Interactions Between WDR44 and Rab11

Project description:DR44 is a Rab11 effector protein that is thought to regulate ciliogenesis by competing with pro-ciliogenesis factors for Rab11 binding. The structure of WDR44 and the molecular mechanism of its interaction with Rab11 is unknown. To explore the interactions between these two proteins multiple WDR44 constructs were designed and pulled down with Rab11. Using the results of the pulldown assay as a guide we used protein complex prediction software, and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify the binding interface between WDR44 and Rab11. Mutagenesis was used to make specific mutations in the putative Rab11 binding region of WDR44.

2023-03-11 | PXD035741 | Pride

HDX analysis of PI3K oncogenic mutants

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.

2023-02-22 | PXD031080 | Pride