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. 2012 Feb 3;11(2):1027-41.
doi: 10.1021/pr200775j. Epub 2012 Jan 9.

In Vivo Application of Photocleavable Protein Interaction Reporter Technology

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Free PMC article

In Vivo Application of Photocleavable Protein Interaction Reporter Technology

Li Yang et al. J Proteome Res. .
Free PMC article

Abstract

In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to Escherichia coli cells, and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating that many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo intercross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology.

Figures

Figure 1
Figure 1. Structure of new generation of pcPIR cross-linker, cross-linking reaction and photocleavage reaction
Figure 2
Figure 2. Specific mass relationships between cross-linked parents and released peptides of different cross-linking relationships
Figure 3
Figure 3. General experimental process of the modified pcPIR technology
3a. Experimental scheme of pcPIR technology. 3b. Four-step LC-mass spectrometric identification-and-verification of the cross-links.
Figure 3
Figure 3. General experimental process of the modified pcPIR technology
3a. Experimental scheme of pcPIR technology. 3b. Four-step LC-mass spectrometric identification-and-verification of the cross-links.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 4
Figure 4. An inter-cross-link example from tryptophanase/L-cysteine desulfhydrase (tnaA)
a. Comparison between laser on and laser off scans: the cross-linked parent peak decreased from laser off to laser on; the released peptides and reporter were generated in the laser on scan; and the sum of neutral masses of released peptides and reporter equals the mass of cross-linked parent with an error of 0.5 ppm. b. Extracted Ion Chromatograms (EICs) of the tnaA cross-linking related products: cross-linked parent, released peptides and the reporter. c. Comparison of EICs between an identified tnaA cross-linked peptide and a non-cross-linked standard peptide Angiotensin from a separated LC-MS/MS experiment. d-e. MS/MS fragmentations and sequences of the two released tnnA peptides f. Manual verification of the tnnA cross-linking relationship g. Mapping the intra-protein tnnA cross-link on its crystallography structure (PDB entry 2OQX). The distance between the two lysine side chains is 16.7 Å.
Figure 5
Figure 5. Homodimer inter-cross-link of kdud
a. Inter-cross-linked parent spectrum of kdud peptide pair b. Released peptide spectrum from kdud c. On-line MS/MS and Mascot search result of the kdud released peptide d. Manual verification of the kdud cross-linking relationship e. Alignment of kdud from E. coli and Ga5DH from S.suis. Red box of lysine residues are cross-linked lysine sites. Orange boxed residues are binding sites from UniProt Knowledge Base. Red under lined sequence (including cross-linking site) is the characteristic motif of SDR family. * indicates conserved residue sites,: shows conservative substitution, and . shows semi-conserved sites. f. Kdud monomeric model. The cross-linked lysine is marked red, and the other three residues in the catalytic tetrad Arg104-Ser150-Tyr163-Lys167 are marked yellow. g. Homodimer kdud protein model docked with PDB entry 3CXR and software SymmDock. The two red residues are the two cross-linked lysine. The cross-linking distance resultant from this model structure was found to be 22.3 Å between lysine side chains.
Figure 5
Figure 5. Homodimer inter-cross-link of kdud
a. Inter-cross-linked parent spectrum of kdud peptide pair b. Released peptide spectrum from kdud c. On-line MS/MS and Mascot search result of the kdud released peptide d. Manual verification of the kdud cross-linking relationship e. Alignment of kdud from E. coli and Ga5DH from S.suis. Red box of lysine residues are cross-linked lysine sites. Orange boxed residues are binding sites from UniProt Knowledge Base. Red under lined sequence (including cross-linking site) is the characteristic motif of SDR family. * indicates conserved residue sites,: shows conservative substitution, and . shows semi-conserved sites. f. Kdud monomeric model. The cross-linked lysine is marked red, and the other three residues in the catalytic tetrad Arg104-Ser150-Tyr163-Lys167 are marked yellow. g. Homodimer kdud protein model docked with PDB entry 3CXR and software SymmDock. The two red residues are the two cross-linked lysine. The cross-linking distance resultant from this model structure was found to be 22.3 Å between lysine side chains.
Figure 5
Figure 5. Homodimer inter-cross-link of kdud
a. Inter-cross-linked parent spectrum of kdud peptide pair b. Released peptide spectrum from kdud c. On-line MS/MS and Mascot search result of the kdud released peptide d. Manual verification of the kdud cross-linking relationship e. Alignment of kdud from E. coli and Ga5DH from S.suis. Red box of lysine residues are cross-linked lysine sites. Orange boxed residues are binding sites from UniProt Knowledge Base. Red under lined sequence (including cross-linking site) is the characteristic motif of SDR family. * indicates conserved residue sites,: shows conservative substitution, and . shows semi-conserved sites. f. Kdud monomeric model. The cross-linked lysine is marked red, and the other three residues in the catalytic tetrad Arg104-Ser150-Tyr163-Lys167 are marked yellow. g. Homodimer kdud protein model docked with PDB entry 3CXR and software SymmDock. The two red residues are the two cross-linked lysine. The cross-linking distance resultant from this model structure was found to be 22.3 Å between lysine side chains.
Figure 5
Figure 5. Homodimer inter-cross-link of kdud
a. Inter-cross-linked parent spectrum of kdud peptide pair b. Released peptide spectrum from kdud c. On-line MS/MS and Mascot search result of the kdud released peptide d. Manual verification of the kdud cross-linking relationship e. Alignment of kdud from E. coli and Ga5DH from S.suis. Red box of lysine residues are cross-linked lysine sites. Orange boxed residues are binding sites from UniProt Knowledge Base. Red under lined sequence (including cross-linking site) is the characteristic motif of SDR family. * indicates conserved residue sites,: shows conservative substitution, and . shows semi-conserved sites. f. Kdud monomeric model. The cross-linked lysine is marked red, and the other three residues in the catalytic tetrad Arg104-Ser150-Tyr163-Lys167 are marked yellow. g. Homodimer kdud protein model docked with PDB entry 3CXR and software SymmDock. The two red residues are the two cross-linked lysine. The cross-linking distance resultant from this model structure was found to be 22.3 Å between lysine side chains.
Figure 5
Figure 5. Homodimer inter-cross-link of kdud
a. Inter-cross-linked parent spectrum of kdud peptide pair b. Released peptide spectrum from kdud c. On-line MS/MS and Mascot search result of the kdud released peptide d. Manual verification of the kdud cross-linking relationship e. Alignment of kdud from E. coli and Ga5DH from S.suis. Red box of lysine residues are cross-linked lysine sites. Orange boxed residues are binding sites from UniProt Knowledge Base. Red under lined sequence (including cross-linking site) is the characteristic motif of SDR family. * indicates conserved residue sites,: shows conservative substitution, and . shows semi-conserved sites. f. Kdud monomeric model. The cross-linked lysine is marked red, and the other three residues in the catalytic tetrad Arg104-Ser150-Tyr163-Lys167 are marked yellow. g. Homodimer kdud protein model docked with PDB entry 3CXR and software SymmDock. The two red residues are the two cross-linked lysine. The cross-linking distance resultant from this model structure was found to be 22.3 Å between lysine side chains.
Figure 6
Figure 6. Homodimer inter-cross-link of GAPDH
a. Inter-cross-linked parent spectrum of GAPDH peptide pair b. Released peptide spectrum from GAPDH c. On-line MS/MS and Mascot search result of the GAPDH released peptide d. Manual verification of the GAPDH cross-linking relationship e. GAPDH tetramer complex crystallography structure and the homodimer cross-link f. The cross-linked lysine residues are marked red and the other NAD+ binding sites are marked yellow
Figure 6
Figure 6. Homodimer inter-cross-link of GAPDH
a. Inter-cross-linked parent spectrum of GAPDH peptide pair b. Released peptide spectrum from GAPDH c. On-line MS/MS and Mascot search result of the GAPDH released peptide d. Manual verification of the GAPDH cross-linking relationship e. GAPDH tetramer complex crystallography structure and the homodimer cross-link f. The cross-linked lysine residues are marked red and the other NAD+ binding sites are marked yellow
Figure 6
Figure 6. Homodimer inter-cross-link of GAPDH
a. Inter-cross-linked parent spectrum of GAPDH peptide pair b. Released peptide spectrum from GAPDH c. On-line MS/MS and Mascot search result of the GAPDH released peptide d. Manual verification of the GAPDH cross-linking relationship e. GAPDH tetramer complex crystallography structure and the homodimer cross-link f. The cross-linked lysine residues are marked red and the other NAD+ binding sites are marked yellow
Figure 6
Figure 6. Homodimer inter-cross-link of GAPDH
a. Inter-cross-linked parent spectrum of GAPDH peptide pair b. Released peptide spectrum from GAPDH c. On-line MS/MS and Mascot search result of the GAPDH released peptide d. Manual verification of the GAPDH cross-linking relationship e. GAPDH tetramer complex crystallography structure and the homodimer cross-link f. The cross-linked lysine residues are marked red and the other NAD+ binding sites are marked yellow

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