Intra- and inter-monomeric crosslinking distinguished by mixed isotope labeling.

<p>MS-spectra obtained by crosslinking of ¹⁴N/¹⁵N oligomers of Hsp21 showing (A) a crosslink formed within the monomers resulting in two peaks with ¹⁴N-¹⁴N and ¹⁵N-¹⁵N (designated as intra-monomeric (<i>intra</i>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone-0038927-t001" target="_blank">Table 1</a>) and (B) a crosslink formed between the monomers resulting in a four peak pattern with ¹⁴N-¹⁴N, ¹⁴N-¹⁵N, ¹⁵N-¹⁴N and ¹⁵N-¹⁵N (designated as inter-monomeric (<i>inter</i>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone-0038927-t001" target="_blank">Table 1</a>). The inter-monomeric crosslinks can originate from crosslinking within or between the oligomers. The two-peak pattern with ¹⁴N-¹⁴N and ¹⁵N-¹⁵N could in principle also originate from inter-monomeric intra-dimeric crosslinking since stable dimers may be the exchanging subunits <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Painter1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-VanMontfort1" target="_blank">[26]</a>.</p

The P₃-R crosslink in the proposed subunit organization of the ClpP₃/R complex.

<p>The identified crosslink between peptide A₁₆₄KEVLANK₁₇₁ of subunit R and peptide I₁₆₉EKDTDR₁₇₅ of subunit P₃ fits the proposed structure model of the Clp proteolytic core complex. A: <i>E. coli</i> ClpP shown in gray (PDB ID 1TYF), with one R (in red) and one P3 (in blue) <i>Synechococcus</i> subunit superimposed on two <i>E. coli</i> ClpP subunits. B: The distance between the Cα atoms of the crosslinked lysine residues is 19 Å in this model, which is compatible with the length of the crosslinker. The figures were prepared with PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). The homology models of ClpR (UniProtKB Q9L4P4) and ClpP3 (UniProtKB Q9L4P3) were downloaded from ‘The Protein Model Portal’ (<a href="http://www.proteinmodelportal.org" target="_blank">www.proteinmodelportal.org</a>), with the models based on templates 1tyfC (residues 24–217) and 1tyfA (residues15–197). They were superimposed onto chains C and D of <i>E. coli</i> ClpP with the ‘magic fit’ function in Swiss-PdbViewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Guex1" target="_blank">[27]</a>.</p

The number of possible crosslinked peptides increasing with sequence length.

<p>The number of theoretically possible peak masses of tryptic unmodified peptides and of crosslinked peptides between 600 and 5000 Da was calculated using the software GPMAW. Data shown from left to right for a single chaperone protein here investigated, Hsp21 (UniProtKB P31170; first 44 amino acids replaced with start methionine, sequence length 184), and a chaperone model substrate protein used in ongoing crosslinking experiments MDH, (P00346, sequence length 338), and two AAA protein complexes. The AAA protein magnesium chelatase Bch IDH complex from <i>Rhodobacter capsulatus</i> is composed of subunit I (P26239, and C-terminal hexa-His, sequence length 356, 10 lysines), subunit D (P26175, and N-terminal hexa-His, sequence length 567, 15 lysines), and subunit H (P26162, and N-terminal hexa-His, sequence length 1195, 44 lysines). The AAA protein Clp-protease from <i>Synechococcus sp.</i> is composed of subunit P (Q9L4P3, and C-terminal hexa-His tag, sequence length 205, 9 lysines), subunit R (Q9L4P4, sequence length 228, 7 lysines), and subunit C (Q55023, sequence length 824, 52 lysines).</p

Crosslinks detected by FINDX map into the protein structure.

<p>Crosslinks detected by FINDX and validated by MSMS are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone-0038927-t001" target="_blank">Table 1</a> and here mapped into the structure of the Hsp21 protein between the Cα atoms of two lysines. Assuming a maximum distance between the two Cα atoms of crosslinkable lysines of 20 Å (12+8 Å  =  length of the BS3 crosslinker and 2 x the lysine side chain), all identified intra-dimeric crosslinks are reconcilable with the dimer structure. The Hsp21 protein is composed of six dimers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Ahrman1" target="_blank">[16]</a> but for clarity only one dimer is presented with subunit A (to the left, dark grey) and subunit B (to the right, lighter grey). Two crosslinks that are inter-monomeric involve the lysine residue K126, which is located in the flexible loop with strand β6, that stabilizes the dimers via strand exchange between the two monomers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Basha1" target="_blank">[24]</a>. The image of the Hsp21 structure model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Lambert2" target="_blank">[19]</a> was prepared with PyMOL (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>).</p

Crosslinks detected by FINDX within a single protein using the crosslinker BS³.

<p>Crosslinking was performed with Hsp21, an oligomeric chaperone small heat shock protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Lambert2" target="_blank">[19]</a>, at a protein concentration of 50 µM. The data shown in the table is the sum of crosslinks observed in three independent experiments with crosslinker to protein ratios of 1∶1, 10∶1 and 50∶1. Only crosslinks of type 2 are shown (not type 0 and 1). Crosslinks were detected with LC-MALDI-TOFTOF by first using the MS-data for screening in FINDX, and subsequent validation of all crosslinks by MSMS.</p>a<p>Crosslinked lysine residues, designated with amino acid number in sequence (M1 refers to crosslinking with the primary amine in the N-terminal residue, methionine).</p>b<p>The distances between the crosslinked lysine residues in the three-dimensional structure model of Hsp21 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone.0038927-Lambert2" target="_blank">[19]</a> are below 20 Å and the cross-links reconcilable with the 3D-structure, m  =  within monomer, d  =  within dimer, (x)  =  one distance was not <20 Å, presumably a crosslink between subunits or oligomers as suggested by its inter-monomeric status in the right-most column, Unstr.  =  crosslinks involving either M1 or lysine residues in the unstructured N-terminal domain (residues 1–81 in sequence) for which the distance cannot be determined.</p>c<p><i>intra</i> and <i>inter</i>  =  intra- and inter-monomeric, -  =  crosslink not examined by ¹⁴N/¹⁵N mixed isotope crosslinking as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone-0038927-g004" target="_blank">Fig. 4</a>.</p

Crosslinking with varying protein concentration and protein-to-crosslinker-ratio.

<p>Crosslinking of the dodecameric Hsp21 protein at varying protein concentrations (5, 10, 50 and 70 µM) and crosslinker to protein molar ratio (1∶1, 1∶10, 1∶50) was evaluated by SDS-PAGE. To each lane, 1.2 µg crosslinked protein was loaded (except for the 70 µM samples where 2.4 µg was loaded) and the gel was stained with CBB.</p

Mass spectra for crosslinks identified in protein multisubunit complexes.

<p>The Clp-protease multisubunit protein complex was crosslinked and the tryptic digest was separated into 192 fractions on the MALDI-target plate. The MS-data were analyzed in FINDX with search restrictions on the isotope doublet peak mass difference accuracy (7 ppm) and intensities (intensity parameter set to 0.22) and matched against >25000 theoretically possible peak masses. Only a few (12) peaks were suggested to be crosslinks, and subjected to MSMS. (A) MS-spectrum showing typical isotope doublet peaks recognized by FINDX. This crosslinked peptide has a theoretical mass MH⁺1886/1898 Da for H12/D12, and corresponds to the crosslink between subunit P₃ peptide I₁₆₉EKDTDR₁₇₅ and subunit R peptide A₁₆₄KEVLANK₁₇₁ and was detected only in 1 out of 192 wells on the MALDI target plate. (B) MSMS-spectrum acquired for crosslink validation. All the fragments containing the crosslinker (AKEx, IEKDx, KDTDRx, EKDTDx) appear as doublet peaks, further confirming the identification.</p

Isotope doublet peaks eluting with D12 before H12.

<p>MS-spectra showing typical elution pattern for a crosslinked peptide in three consecutive LC-fractions (A, B, C) in a 192-well LC-separation. This crosslinked peptide has a theoretical mass MH⁺1712/1724 Da for H12/D12, and corresponds to the crosslink between T₁₇₂KVER₁₇₆ and K₁₇₇VIDVQIQ₁₈₄ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038927#pone-0038927-t001" target="_blank">Table 1</a>).</p