Dataset Information

Crosslinking/mass spectrometry analysis of Human Separase-Scc1-fusion and cohesin SMC1/SMC3 complexes

ABSTRACT: In this study, we employed the photoactivatable crosslinker (sulfo-SDA) to investigate protein-protein interaction between human Separase-Scc1 fusion protein and SMC1/SMC3 cohesin subunits complex in the presence of DNA.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kitaik Lee

LAB HEAD: Francis J. O'Reilly

PROVIDER: PXD061667 | Pride | 2025-08-28

REPOSITORIES: Pride

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			Action	DRS
	E241109_04_KL_Boland_Separase_Scc1_Cohesin_SulfoSDA_pepSEC3_FullMS1.raw	Raw
	E241109_05_KL_Boland_Separase_Scc1_Cohesin_SulfoSDA_pepSEC4_FullMS1.raw	Raw
	E241109_06_KL_Boland_Separase_Scc1_Cohesin_SulfoSDA_pepSEC5_FullMS1.raw	Raw
	E241109_07_KL_Boland_Separase_Scc1_Cohesin_SulfoSDA_pepSEC6_FullMS1.raw	Raw
	E241109_09_KL_Boland_Separase_Scc1_Cohesin_SulfoSDA_pepSEC7_FullMS1.raw	Raw

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Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.

Project description:The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic, i.e. greater than additive, ability to inhibit the growth of Listeria monocytogenes. Full genome microarrays were used to investigate the synergistic transcriptomic response of two L. monocytogenes strains, h7858 (serotype 4b) and f6854 (serotype 1/2a), to organic acid, under conditions controlling for osmotic and cold stress. Strains were exposed to BHI broth at 7°C with 4.65% water phase (w.p.) NaCl at pH 6.1 treated with 2% w.p. potassium lactate, 0.14% w.p. sodium diacetate, the combination of both at the same levels, or no inhibitors as control. RNA was extracted 8h after exposure, during lag phase, to capture gene expression changes during adaptation to the organic acid stress. Treatment with organic acids induced massive global transcriptional changes, with 1041 and 640 genes differentially expressed in h7858 and f6854. Major effects of treatment with lactate and diacetate are (i) a total of 474 and 209 genes, for h7858 and f6854, that showed synergistic expression differences, (ii) differential expression of membrane ion transport genes including those encoding ABC transporters of metals and decreased multi-drug transporter expression many ABC, PTS, and drug transporter systems, including increased PTS sugar transport and decreased multi-drug transporter expression, and (iii) altered metabolism including induction of a nutrient limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of lactate and acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional synergies in L. monocytogenes growth inhibition could be achieved by treatments that interfere with cellular energy generation processes. The dye-swapped, single loop design hybridizes a single biological replicate of both 2% water phase lactate (PL) and 0.14% water phase acetate (SDA) treatments to both the control (CTRL) and combination (PLSDA) treatments (4 hybridizations), using opposite dye labels for each sample, and a second biological replicate is hybridized with dye assignments swapped (4 more hybridizations) to balance labeling effects. The design was repeated twice comprising 16 hybridizations over 4 biological replicates for each strain, and 32 total hybridizations over both h7858 and f6854.

Dataset Information

Crosslinking/mass spectrometry analysis of Human Separase-Scc1-fusion and cohesin SMC1/SMC3 complexes

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