Table 1.
PDE6 holoenzyme intra- and intermolecular cross-linked peptides
Cross-linked peptides were identified following chemical cross-linking of 10–50 pmol of purified rod PDE6 holoenzyme as described under “Experimental procedures.” Except where indicated with superscripts, samples consisted of native PDE6 holoenzyme and were digested with trypsin prior to mass spectrometric analysis. Exp. m/z is the experimentally measured mass-to-charge ratio, z is the charge state of the peptide, and Δ is the accuracy measured in parts per million. The cross-linked peptides are defined as the protein subunit (pep1 and pep2) and amino acid residue number (aa1 and aa2) identified using the indicated cross-linker. In the aa1 column, the presence of a single-letter amino acid residue preceding the residue number indicates an amino acid substitution of the wild-type Pγ sequence at the site of cross-linking. In addition to the cross-links in this table, the PDE6 structural model included spatial constraints from cross-links reported previously for the PDE6 holoenzyme (20). BMH, 1,6-bismaleimidohexane; BMOE, bis-maleimidoethane; BS(PEG)9, PEGylated bis(sulfosuccinimidyl)suberate; Sulfo-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; Sulfo-SDA, sulfosuccinimidyl 4,4′-azipentanoate.
| Exp. m/z | z | Δ | pep1 | aa1 | pep2 | aa2 | Cross-linker |
|---|---|---|---|---|---|---|---|
| ppm | |||||||
| 658.9788 | 3 | 7.4 | Pβ | 471 | Pβ | 475 | EDC |
| 431.8966 | 3 | −3.5 | Pβ | 675 | Pβ | 813 | EDC |
| 431.8969 | 3 | −2.8 | Pβ | 675 | Pβ | 815 | EDC |
| 405.4728 | 4 | 2.9 | Pβ | 823 | Pβ | 832 | EDC |
| 540.2943 | 3 | 2.3 | Pβ | 824 | Pβ | 832 | EDC |
| 606.6677 | 3 | 1.7 | Pβ | 825 | Pβ | 827 | Sulfo-SDA |
| 540.2935 | 3 | 0.81 | Pβ | 826 | Pβ | 828 | EDC |
| 530.9468 | 3 | 2.4 | Pβ | 826 | Pβ | 829 | Sulfo-SDA |
| 530.9463 | 3 | 1.4 | Pβ | 826 | Pβ | 831 | Sulfo-SDA |
| 567.6403 | 3 | −6.7 | Pβ | 826 | Pα/Pβ | 445/444 | Sulfo-SDA |
| 425.9827 | 4 | −5.2 | Pβ | 826 | Pα/Pβ | 442/441 | Sulfo-SDA |
| 606.6667 | 3 | 0.034 | Pβ | 826 | Pβ | 828 | Sulfo-SDA |
| 524.9417 | 3 | −7.8 | Pβ | 826 | Pα/Pβ | 444/443 | Sulfo-SDA |
| 573.6439 | 3 | 0.044 | Pβ | 826 | Pβ | 832 | Sulfo-SDA |
| 578.0868 | 4 | −6.9 | Pγ | 1a | Pβ | 78 | BS3 |
| 921.2301 | 4 | 6.6 | Pγ | C2b | Pβ | 84 | BMH |
| 678.3601 | 3 | −9.4 | Pγ | 4c | Pβ | 146 | EDC |
| 1034.3080 | 4 | 2.5 | Pγ | 7d | Pβ | 184 | BS(PEG)9 |
| 926.7150 | 4 | 0.73 | Pγ | C18e | Pα | 383 | Sulfo-MBS |
| 1113.0730 | 2 | −6.9 | Pγ | C18e | Pβ | 92 | BMH |
| 1027.5500 | 3 | 5.9 | Pγ | C18e | Pα | 233 | BMH |
| 653.0510 | 4 | −1.6 | Pγ | 31a | Pβ | 200 | Sulfo-MBS |
| 596.0509 | 4 | −15 | Pγ | 41c,f | Pα | 469 | Sulfo-SDA |
| 1063.2268 | 3 | −17 | Pγ | 44 | Pα/Pβ | 613/611 | BS3 |
| 800.6560 | 4 | −8.8 | Pγ | 44 | Pβ | 475 | BS3 |
| 498.4935 | 4 | −17 | Pγ | 52c,f | Pα/Pβ | 328/326 | EDC |
| 911.4194 | 3 | −15 | Pγ | K62c,f | Pβ | 450 | EDC |
| 911.4185 | 3 | −4.8 | Pγ | K62c,f | Pβ | 446 | EDC |
| 660.0972 | 4 | −1.3 | Pγ | K62c,d | Pα/Pβ | 394 | EDC |
| 879.7957 | 3 | 0.78 | Pγ | K62c,d | Pα/Pβ | 393 | EDC |
| 478.5120 | 4 | 0.34 | Pγ | K65c,g | Pα | 767 | EDC |
| 670.0686 | 4 | −13 | P | C68g | Pβ | 839 | BMOE |
a Sample consisted of Pαβ reconstituted with recombinant, wildtype rod Pγ.
b Pαβ reconstituted with Pγ2C/68S.
c Trypsin/Asp-N double digest.
d Pαβ reconstituted with Pγ58K/62K/65K/73K.
e Pαβ reconstituted with Pγ18C/68S.
f Pαβ reconstituted with Pγ62K/65K/73K/79K.
g Pαβ reconstituted with Pγ53K/62K/65K/73K.