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Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes

ABSTRACT: Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry (qCLMS). Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II (pol II) complexes.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Jie Luo

LAB HEAD: Jeff Ranish

PROVIDER: PXD056825 | Pride | 2024-11-13

REPOSITORIES: Pride

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			Action	DRS
	ABP-127-inter.xls	Xls
	ABP-127-intra.xls	Xls
	JL033021_040221_RPA2HFH_Q2_10to1_100B.mzXML	Mzxml
	JL033021_040221_RPA2HFH_Q2_10to1_1ug.mzXML	Mzxml
	JL033021_040221_RPA2HFH_Q2_10to1_50B.mzXML	Mzxml

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Project description:The HOP2/MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes, and promotes the repair of DNA double strand breaks. The HOP2/MND1 dimer forms via the central, split coiled coils (CC1 and CC2) present in both proteins, and the resulting complex contains several flexible regions such as the hinge between the coils. To investigate the conformational flexibility of the heterodimer, important for understanding mechanistic details of HOP2/MND1 function, we studied the spatial relation of the complex partners and their domains in solution. We performed chemical cross-linking in combination with mass spectrometry (XL-MS) to generate distance restraints, which were subsequently used for molecular modeling. The final XL-MS workflow encompassed the use of cross-linkers with varying spacer arm lengths, quenching, digestion, size exclusion enrichment and HCD based LC-MS/MS detection prior to data evaluation. We applied and systematically tested two different homobifunctional amine-reactive crosslinkers (DSS-11.4 Å and BS2G 7.7 Å) and one zero-length heterobifunctional crosslinker (EDC). Crosslinked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for crosslink guided molecular modeling. Our miniaturized SEC approach reduced the required starting material and led to a high amount of crosslinked peptides allowing the analysis of replicates. The majority of the identified crosslinks was found in the coiled coil domains, indicating a parallel orientation of the interaction partners. Furthermore, flexibility of the C-terminal head domains of HOP2 and MND1 was observed. The experimentally derived distance constraints combined with an iterative comparative modeling approach not only confirm the elongated, open conformation predicted by the crystal structure of the Giardia lamblia Hop2-Mnd1 heterodimer, but further suggest the coexistence of a closed complex conformation in solution.

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Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes

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