The isobaric Qlinker quantitative 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 quantify the relative abundance of crosslinks and monolinks derived from Q2linker modification of affinity purified pol I at designated ratios. Example spectra of a monolinked peptide (b) and a crosslinked peptide (c) with the observed reporter ion intensities at each mixing ratio. d) Observed vs. expected log2 (127/126) reporter ion ratios for monolinks. e) Observed vs. expected log2 (127/126) reporter ion ratios for crosslinks. Boxplot in R was used to create the graphs in d) and e). Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.

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, maltose-bound conformation (brown, 1n3w.pdb) are shown on the left. A close-up view of the “balancing interface” is shown on the right. In the closed conformation, the sequence between amino acids 301 to 312 forms an alpha helix (gray) and K313 forms a salt bridge with E310. The helix and the salt bridge are disrupted in the open conformation (cyan). A crosslinker swapping experiment was performed on the same preparation of MBP. b) Quantification of monolinked and crosslinked peptides involving K78 and K95 from apo-CaM and CaM with Ca2+ and CBP. Structures of apo-CaM (left) and CaM + CBP (blue) + Ca2+ (right) are shown with key lysine residues space filled and magenta.

Q2linkers detect structural changes during TFIIA/TBP/TFIIB ternary complex formation.

a and 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 binary complexes (126). The x-axis is the ranking of the log2 (127/126) ratios. Crosslinks with the highest ratios are labeled. c) The structure of TFIIA, TBP, and TFIIB in the pol II PIC complex (5fmf) is shown. TFIIB is rainbow colored with blue at the N-terminus and red at the C-terminus. Residues in the central region of TFIIB that are involved in crosslinks with high ratios are shown as space filled. The regions of TFIIB that interact with the Rpb1 clamp and Rpb2 protrusion are also shown.

Q2linkers detect conformational changes in large protein complexes

a) The SDS-PAGE gel of affinity purified pol II from WT and ΔRpb4 strains and experimental design for studying conformational changes in RNA polymerase II due to deletion of Rpb4. b-d) The log2(126/127) ratio comparisons in experiment I and II for holo-Pol II and ΔRpb4-pol II. x-axis is ΔRpb4-pol II (126)/holo-pol II (127). y-axis is holo-pol II (126)/ΔRpb4-pol II (127). Enrichment in the holo-pol II sample (labelled with C2q2 in experiment I) will have a smaller log2(126/127) ratio on the x-axis; enrichment in the holo-pol II (labelled with C1q2 in experiment II) will have a higher log2(126/127) ratio on the y-axis. Each green dot corresponds to one unique pair of crosslinking sites identified in both experiments. Each blue dot on the x-axis corresponds to one unique pair of crosslinking sites identified only in experiment I and each yellow dot on the y-axis corresponds to one unique pair of crosslinking sites identified only in experiment II. Only the green dots are used for the linear regression analysis. b) interlinks, c) intralinks and d) monolinks. Dashed lines indicate log2 ratio = 1. The same preparation of each protein complex was used in a crosslinker swapping experiment.

Q2linkers detect conformational changes in pol II complexes.

a) Switch 2 (Rpb1:328-346) conformational changes in ΔRpb4-pol II (cyan) and holo-pol II (gray). The crosslinked lysine residues are shown as spheres. Cα− Cα distances between crosslinked lysines are indicated b) Structural comparison of crosslinks involving switch 5 (red, Rpb1:1431-1433) for ΔRpb4-pol II (cyan, left), holo-pol II (gray, right) and the merged structures (middle). c) Structural comparison of the region of Rpb5 involving the crosslink between K161 and K171 in ΔRpb4-pol II and holo-pol II. A salt bridge (not shown) is formed between K161 and E172 in both structures. d) Structure of Rpb1 near the crosslink between Rpb1:K689 and Rpb1:K728 in ΔRpb4-pol II and holo-pol II. In all structures, ΔRpb4-pol II is cyan and holo-pol II is gray. NZ−NZ distances between crosslinked lysines are indicated.