Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes
- Institute for Systems Biology, United States
Figures
Figure 1 with 4 supplements
The isobaric Qlinker quantitative crosslinking-mass spectrometry (CLMS) approach for studying conformational and structural changes in proteins and protein complexes.
(a) The structure of Q2linkers and the general scheme for qCLMS using Q2linkers. The 13C atom in C1q2 or C2q2 is indicated by red font. (b–e) Experiment to evaluate the ability of Q2linkers to …
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Figure 1—source data 1
Table containing information about the monolinks reported in Figure 1b and d.
The information includes the peptide sequence and protein name, site of Qlinker modification, score, reporter ion intensities, and reporter ion ratios.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig1-data1-v1.xlsx
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Figure 1—source data 2
Table containing information about the intralinks reported in Figure 1c and e.
The information includes the peptide sequences and protein names, sites of Qlinker modification, score, search engine (N = Nexus, P = pLink2), reporter ion intensities, and reporter ion ratios.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig1-data2-v1.xlsx
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Figure 1—source data 3
Table containing information about the interlinks reported in Figure 1c and e.
The information includes the peptide sequences and protein names, sites of Qlinker modification, score, search engine (N = Nexus, P = pLink2), reporter ion intensities, and reporter ion ratios.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
Synthesis of Q2linkers.
The 13C atom in C1q2 or C2q2 is indicated by red font.
Figure 1—figure supplement 2
Experimental procedure to evaluate the ability of Q2linkers to quantify the relative abundances of crosslinks and monolinks derived from Q2linker modification of affinity-purified pol I.
M = molecular weight markers.
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Figure 1—figure supplement 2—source data 1
File containing the original gel for Figure 1—figure supplement 2, indicating the relevant bands and molecular weight markers.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original file for the gel displayed in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig1-figsupp2-data2-v1.zip
Figure 1—figure supplement 3
Observed vs. expected log2 (126/127) reporter ion ratios for individual monolinks (A) and crosslinks (B) at different mixing ratios.
There are 298 monolinks and 32 crosslinks identified in all runs.
Figure 1—figure supplement 4
The ratio distributions for interlinks, intralinks, and monolinks from the pol II +/-a-amanitin experiment.
Figure 2
Q2linkers detect conformational changes in protein biosensors.
(a) Quantification of a monolinked peptide from maltose binding protein (MBP) with or without maltose. Structures of MBP in the open, ligand-free conformation (green, 1mpb.pdb) and the closed, …
Figure 3
Q2linkers detect structural changes during TFIIA/TBP/TFIIB ternary complex formation.
(a, b) The distribution of log2 (127/126) ratios for interlinks (a) and intralinks (b) from the Qlinker experiment comparing the TFIIA/TBP/TFIIB ternary complex (127) to the TFIIA/TBP and TFIIB/TBP …
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Figure 3—source data 1
Table containing information about the interlinks reported in Figure 3a.
The information includes the peptide sequences and protein names, sites of Qlinker modification, score, search engine (N = Nexus, P = pLink2), reporter ion intensities, and reporter ion ratios.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig3-data1-v1.xlsx
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Figure 3—source data 2
Table containing information about the intralinks reported in Figure 3b.
The information includes the peptide sequences and protein names, sites of Qlinker modification, score, search engine (N = Nexus, P = pLink2), reporter ion intensities, and reporter ion ratios.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig3-data2-v1.xlsx
Figure 4 with 1 supplement
Q2linkers detect conformational changes in large protein complexes.
(a) The SDS-PAGE gel of affinity-purified pol II from WT and ∆Rpb4 strains and experimental design for studying conformational changes in RNA polymerase II due to deletion of Rpb4. (b–d) The …
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Figure 4—source data 1
File containing the original gel for Figure 4a, indicating the relevant bands and molecular weight markers.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig4-data1-v1.zip
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Figure 4—source data 2
Original file for the gel displayed in Figure 4a.
- https://cdn.elifesciences.org/articles/99809/elife-99809-fig4-data2-v1.zip
Figure 4—figure supplement 1
Structural comparison of holo-pol II (5u5q) and core-pol II (li3q).
Core-pol II without Rpb4/Rpb7 is colored brown and holo-pol II is colored gray with Rpb4 and Rpb7 in light blue. The five switches that cause the movement of the clamp are colored in magenta.
Figure 5
Q2linkers detect conformational changes in pol II complexes.
(a) Switch 2 (Rpb1:328–346) conformational changes in ∆Rpb4-pol II (cyan) and holo-pol II (gray). The crosslinked lysine residues are shown as spheres. Cα−Cα distances between crosslinked lysines …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3)-CodonPlus-RIL | Agilent | Cat# 230245 | Chemically Competent cells |
| Strain, strain background (Saccharomyces cerevisiae) | BY4741 with C-terminal His6-3XFLAG-His6-Ura3 (HFH) tag on RPA2 | This paper | Available on request from the Ranish lab | |
| Strain, strain background (S. cerevisiae) | BY4741 with C-terminal (HFH) tag on RPB3 | This paper | Available on request from the Ranish lab | |
| Strain, strain background (S. cerevisiae) | Δrpb4 strain with C-terminal (HFH) tag on RPB2 | This paper | Available on request from the Ranish lab | |
| Recombinant DNA reagent | His6-tagged TBP | Dr. Steven Hahn (Fred Hutchinson Cancer Research Center) | ||
| Recombinant DNA reagent | His6-tagged TFIIB | Dr. Steven Hahn (Fred Hutchinson Cancer Research Center) | ||
| Recombinant DNA reagent | His6-tagged TFIIA | PMID:28259734 | ||
| Peptide, recombinant protein | Human Brain Calmodulin | MilliporeSigma | Cat# 208698500ug | |
| Peptide, recombinant protein | Calmodulin binding peptide 1 | GenScript Biotech Corp. | Cat# RP13247 | |
| Peptide, recombinant protein | Bacterial maltose binding protein | Novus Biologicals | Cat# NBC118538 | |
| Chemical compound, drug | Di-tert-butyl 3,3'-Iminodipropionate | TCI America | D4110 | CAS: 128988-04-5 |
| Chemical compound, drug | Bromoacetic acid (1-13C) | Cambridge Isotope Laboratories, Inc | CLM-723-PK | CAS: 57858-24-9 |
| Chemical compound, drug | Bromoacetic acid (2-13C) | Cambridge Isotope Laboratories, Inc | CLM-724-PK | CAS: 64891-77-6 |
| Chemical compound, drug | cis-2’–6’-Dimethylpiperidine | MilliporeSigma | D180300 | CAS: 766-17-6 |
| Software, algorithm | Nexus | PMID:30343899 | Source code 1 | |
| Software, algorithm | pLink2 | PMID:31363125 | ||
| Software, algorithm | Rawconverter | PMID:26499134 | ||
| Software, algorithm | Trans-Proteomics Pipeline (TPP)/ | PMID:36648445 | http://tools.proteomecenter.org/wiki/index.php?title=Software:TPP |
Additional files
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Supplementary file 1
Table containing information about the interlinks, intralinks, and monolinks reported in Figure 4 .
The information includes the peptide sequences and protein names, sites of Qlinker modification, score, search engine (N = Nexus, P = pLink2), reporter ion intensities, and reporter ion ratios.
- https://cdn.elifesciences.org/articles/99809/elife-99809-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/99809/elife-99809-mdarchecklist1-v1.docx
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Source code 1
Nexus algorithm.
- https://cdn.elifesciences.org/articles/99809/elife-99809-code1-v1.zip
