A targeted releasable affinity probe (TRAP) for in vivo photocrosslinking.
Protein crosslinking, especially coupled to mass-spectrometric identification, is increasingly used to determine protein binding partners and protein-protein interfaces for isolated protein complexes. The modification of crosslinkers to permit their targeted use in living cells is of considerable importance for studying protein-interaction networks, which are commonly modulated through weak interactions that are formed transiently to permit rapid cellular response to environmental changes. We have therefore synthesized a targeted and releasable affinity probe (TRAP) consisting of a biarsenical fluorescein linked to benzophenone that binds to a tetracysteine sequence in a protein engineered for specific labeling. Here, the utility of TRAP for capturing protein binding partners upon photoactivation of the benzophenone moiety has been demonstrated in living bacteria and mammalian cells. In addition, ligand exchange of the arsenic-sulfur bonds between TRAP and the tetracysteine sequence to added dithiols results in fluorophore transfer to the crosslinked binding partner. In isolated protein complexes, this release from the original binding site permits the identification of the proximal binding interface through mass spectrometric fragmentation and computational sequence identification.
Yan
Ping
P
Novozymes, Inc., 1445 Drew Ave, Davis, CA 95618, USA.
Wang
Ting
T
Newton
Gregory J
GJ
Knyushko
Tatyana V
TV
Xiong
Yijia
Y
Bigelow
Diana J
DJ
Squier
Thomas C
TC
Mayer
M Uljana
MU
D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D000345:Affinity Labels; D002147:Calmodulin; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011506:Proteins; C085606:SlyD protein, E coli; D009219:Myosin-Light-Chain Kinase; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D019696:Peptidylprolyl Isomerase