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Hydrogen exchange mass spectrometry analysis of the Spc72-Stu2 complex

ABSTRACT: To verify the Stu2-Spc72 complex predicted by AlphaFold2 hydrogen exchange mass spectrometry was performed.

INSTRUMENT(S):

ORGANISM(S): Candida Albicans (yeast)

SUBMITTER: Marcin Luzarowski

LAB HEAD: Marcin Luzarowski

PROVIDER: PXD057184 | Pride | 2025-01-07

REPOSITORIES: Pride

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			Action	DRS
	100_Spc72_1_01_3209.d.zip	Other
	100_Spc72_1_01_3239.d.zip	Other
	100_Stu2_1_01_3214.d.zip	Other
	100_Stu2_1_01_3244.d.zip	Other
	30sec_Spc72_1_01_3213.d.zip	Other

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Publications

Structural insights into the interplay between microtubule polymerases, γ-tubulin complexes and their receptors.

Zheng Anjun A Vermeulen Bram J A BJA Würtz Martin M Neuner Annett A Lübbehusen Nicole N Mayer Matthias P MP Schiebel Elmar E Pfeffer Stefan S

Nature communications 20250105 1

The γ-tubulin ring complex (γ-TuRC) is a structural template for controlled nucleation of microtubules from α/β-tubulin heterodimers. At the cytoplasmic side of the yeast spindle pole body, the CM1-containing receptor protein Spc72 promotes γ-TuRC assembly from seven γ-tubulin small complexes (γ-TuSCs) and recruits the microtubule polymerase Stu2, yet their molecular interplay remains unclear. Here, we determine the cryo-EM structure of the Candida albicans cytoplasmic nucleation unit at 3.6 Å r ...[more]

PMID: 39757296

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HX-MS data: Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA

Project description:The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of HSF1 activity regulation remains poorly understood at a molecular level. In metazoa Hsf1 trimerizes upon heat shock through a leucin-zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucinezipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation Hsc70 first binds to a high affinity site in the transactivation domain leading to partial attenuation of the response and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.

2020-06-22 | PXD017406 | Pride

Substrate-induced conformational dynamics of the dopamine transporter

Project description:The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.

2019-11-12 | PXD013841 | Pride

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Project description:We developed an HX-MS2 strategy that supports the acquisition of CID-generated fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining the rich set of deuterated fragments, using the extra dimension of data to simultaneously confirm the peptide ID and authenticate (even correct) MS1-based deuteration calculations. The high redundancy provided by the fragments generates an opportunity to determine the confidence of deuterium calculations using a combinatorial strategy. The approach requires DIA-based acquisition methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Considerable time is saved through auto-validation and more importantly, more complex samples can now be characterized and at higher throughput, as illustrated in a drug binding analysis of the ultralarge kinase DNA-PKcs, isolated directly from mammalian cells.

2023-09-08 | PXD045012 | Pride

Recommendations for Automating Hydrogen/Deuterium Exchange Mass Spectrometry Measurements using Data-Independent Acquisition Methods

Project description:Two test HX-DIA datasets, Phosphorylase B kinetics and nanobody binding, that can be used to explore the features of DIA-HX and the AutoHX software.

2025-01-22 | PXD060028 | Pride

Subzero hydrogen-deuterium exchange CE-MS

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2022-12-22 | PXD034245 | Pride

Transcription profiling of yeast recovery after hydrogen peroxide stress

Project description:Yeast cells response during recovery after hydrogen peroxide stress. Comparison of yeast cells treated with 1.5mM hydrogen peroxide for 30 min with cells allowed to recover for 60 min

2006-01-28 | E-MEXP-326 | biostudies-arrayexpress

The dopamine transporter antiports potassium to increase the uptake of dopamine

Project description:The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we found that K+ increased dopamine uptake by the drosophila dopamine transporter in liposomes, and visualized Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.

2022-05-20 | PXD032663 | Pride

GroEL interaction and folding of MetF studied by hydrogen/deuterium exchange – mass spectrometry

Project description:The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in E. coli by transiently encapsulating non-native substrate in a nano-cage formed by the GroEL ring cavity and the lid-shaped GroES. We analyzed the spontaneous and chaperonin-assisted folding of the essential enzyme 5,10-methylenetetrahydrofolate reductase (MetF) of E. coli, an obligate GroEL/ES substrate. We found that MetF, a homotetramer of 33 kDa subunits with (8) TIM-barrel fold, is unable to fold spontaneously, even in the complete absence of aggregation, and populates a kinetically trapped folding intermediate(s) (MetF-I). GroEL/ES recognized MetF-I and catalyzed its folding with high efficiency, at a half-time of ~4 s and with more than 50% of protein folded in a single round of encapsulation. Analysis by hydrogen/deuterium exchange at peptide resolution showed that the MetF subunit folds to completion in the GroEL/ES nano-cage and binds the co-factor FAD.

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Effect of mifepristone on gene expression in Drosophila

Project description:Illumina sequencing was used to assay the effect of mifepristone treatment on gene expression in adult Drosophila, including males, virgin females and mated females. Males of strain w[1118]; p53B[6] were crossed to virgins of w[1118]; rtTA(3)E2 and progeny males and virgins were collected over 48 hours. One half of the virgins were mated to w[1118] males at ratio of 1:1 virgins to males for 4 days. Mated females were then separated from the w[1118] males. The mated females, males and virgins females were then maintained at approximately 20 flies per vial, on food with and without supplementation with 160ug/ml mifepristone for 12 days. Total fly RNA was isolated from 20 animals per sample. Three replicate samples were generated for each type of flies: males, mated females and virgin females.

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The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA - HX-MS Data

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