Article Figures & Data
Figures
- FIG 1
(A) Sucrose density isolation of CpcG-PBS and CpcL-PBS. (B) SDS-PAGE analysis of CpcG-PBS and CpcL-PBS. (C) Mass spectrometry quantification of protein subunits in CpcG-PBS and CpcL-PBS. In the heat map representation, highlighting the component differences, the relevance of effects is related to a ratio, not to a visual difference. Ratio* (PEAKS label-free quantification) indicates the CpcL-PBS/CpcG-PBS ratio. (D) Absorption spectra of the sucrose gradient bands normalized at the absorption maxima. The spectrum difference (Dif) is the difference spectrum between CpcG-PBS and CpcL-PBS. (E and F) Fluorescence emission spectra of CpcG-PBS and CpcL-PBS upon excitation at 580 nm at room temperature (RT) (E) and at 77 K (F).
- FIG 2
(A) Label-free rod polypeptide quantification of CpcG-PBS and CpcL-PBS. (Error bars indicate standard deviations.) (B and C) Model of a single rod of CpcG-PBS (B) and CpcL-PBS (C). (D) Product-ion (MS/MS) spectrum of CpcG1 protein N-terminal peptide. (Inset) N-terminal polypeptide sequence of CpcG1 (slr2051) and CpcL (sll1471) proteins. (E) Product-ion (MS/MS) spectrum of CpcL N-terminal peptide.
- FIG 3
MS data showing a cross-link between FNRL-K69 and CpcB-M1 (N terminus). (A) The mass spectrum of precursors for the light and heavy cross-linked species (BS3-H12/D12), displaying the isotopic fingerprint of a peak doublet of equal intensity, separated by m/z 4.0251 (z = 3) (B) Product-ion spectrum of the cross-linked peptide with BS3-H12. (C) Product-ion spectrum of the cross-linked peptide with BS3-D12. Ȧ, top peptide; Ӓ, bottom peptide.
- FIG 4
MS data showing a cross-link between FNRL and CpcB. Reference ions without the cross-linker reagent (Ȧy9++ at m/z 469.24, Ȧy9+ at m/z 937.47) and ions with the characteristic 12-Da shift that contain cross-linkers are indicated (Ȧb1+, Ȧb2+, and Ȧb3+). Overall, the product-ion coverage is 71% for y ions and 18% for b ions. The isotopic ion coverage (both y and b ions) is 18%.
- FIG 5
Iterative threading assembly refinement (I-TASSER) method for protein structure prediction of FNRL N-terminal linker domain (FNRL-LD; 89 amino acids). (A to E) Five models are shown with decreasing C-scores (I-TASSER). (F) Bioinformatics analysis of FNRL-LD using the ConSurf server (38, 39, 53), showing the conserved (purple) α helix and β sheet and the variable loop regions and N-terminal flexible domain (teal). (G) Side views of the five models of FNRL-LD. The first methionine of each model is shown as a sphere with color rendering consistent with models in Fig. 1A to E, respectively. (H) Cartoon representation of allophycocyanin core linker (ApcC) complex: ApcA (marine), ApcB (dark blue), and ApcC (purple). (I) Three rounds of alignment of CpcA/B heterodimer (Synechocystis 6803 α and β subunit; PDB entry 4F0T) with ApcA/B (1B33), followed by homology modeling of five predicted FNRL-LD structures with ApcC (PDB entry 1B33).
- FIG 6
(A) View of all amino acids involved in FNRL-LD-PC cross-linking: CpcB-1M (purple), CpcA-1M (blue) from trimeric phycocyanin, K69 (orange), and 1M (green) of FNRL. K69 and 1M from five models are shown. (B) Side view of FNRL-LD (5 models) in PC trimer, highlighting K69 and 1M locations relative to the α-helix and β-sheet of FNRL-LD and the PC trimer. (C) Optimized model (model E in Fig. 5B) with the least spatial conflicts and most favorable cross-linking chemistry. The N-terminal extension region of FNRL adopts an orientation close to the proximal side of CpcA. K69 and M1 of FNRL are located close to N termini of CpcA and CpcB in one heterodimer. (D) Surface representation of trimeric phycocyanin and electrostatic potential surface representation of FNRL-LD. CpcA, wheat; CpcB, lime.
Tables
- TABLE 1
Spatial distance analysis of cross-linked pairs using Xwalka
Model and Xlink FNRL CpcB CpcA Distance (Å) Euclidean Amine SASD RMSD A 1 K69 M1 13.9 11.2 23.8 19.5 2 M1 M1 24.8 23.5 33.1 3 M1 M1 30 27 34.2 B 1 K69 M1 13.6 8.4 22.3 25.9 2 M1 M1 27.2 24.8 44.3 3 M1 M1 32 29.8 39.9 C 1 K69 M1 14.4 9.4 24.1 23.9 2 M1 M1 25 25.6 40.3 3 M1 M1 28.8 28.7 38.3 D 1 K69 M1 14.6 13.5 19.9 20.7 2 M1 M1 24.1 21.3 36.1 3 M1 M1 28.5 25 37.2 E 1 K69 M1 14.1 9.9 21.2 10.7 2 M1 M1 15.3 13.8 22.2 3 M1 M1 18.4 16.4 23.1 ↵a Xwalk (spatial distance) analysis (51) of cross-linking pairs from Fig. 6A. Listed are the Euclidean Cα-Cα distances, the side chain amine groups distances, and SASD (see the text for details) between paired amino acids. Xi, Xwalk calculated value (Å). C = 11.4 Å. RMSD was calculated as
