Dataset Information

Deciphering Diazirine Reaction Mechanism: through Irradiation Modulation for Residue-Specific Distance Restraints in Protein Photo-Crosslinking

ABSTRACT: The diazirine photoreactive group has been widely used for photo-labeling and photo-cross-linking (PXL) of proteins. Yet, due to the mechanistic complexity of diazirine photo-reaction, site-specific and quantitative analysis of protein PXL data remains challenging. Herein, we have built an in-line photo-reaction monitoring system, capable of systematically varying optical power density and irradiation time. Using this system, we have determined the kinetic parameters of diazirine photo-reactions, and shown that an alkyl diazirine undergoes rearrangement upon irradiation to yield a diazo intermediate. A carbene intermediate is produced from the diazo intermediate upon further irradiation, whereas at a relatively small proportion, it is produced directly from the diazirine. Both diazo and carbene intermediates react with protein side-chains. Significantly, the diazo intermediate preferentially reacts with polar side-chains, exhibiting much higher specificity than the carbene intermediate. As the diazo and carbene intermediates are generated sequentially, modulating irradiation time and optical power density can achieve site- or space- selective PXL results through different mechanisms. Together, we have established a new approach for the kinetic mechanism of photo-reactions, and paved the way for quantitative analysis of PXLs to visualize protein structure and dynamics.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: yida jiang

LAB HEAD: Yida Jiang,

PROVIDER: PXD048452 | Pride | 2024-06-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	ADRM1.fasta	Fasta
	LUMOS2_20231216_PKU_TC_JYD_PPASE_H_R1.raw	Raw
	LUMOS2_20231216_PKU_TC_JYD_PPASE_H_R2.raw	Raw
	LUMOS2_20231216_PKU_TC_JYD_PPASE_H_R3.raw	Raw
	LUMOS2_20231216_PKU_TC_JYD_PPASE_H_duplicate_count_combined.xlsx	Xlsx

Items per page:

1 - 5 of 82

Similar Datasets

Project description:Dialkyldiazirines have emerged as a photoaffinity probe of choice for interactome mapping in live cell experiments. Upon irradiation, ‘linear’ dialkyldiazirines produce dialkylcarbenes, which are susceptible to both intramolecular reactions and unimolecular elimination processes, as well as diazoalkanes, which also participate in intermolecular labeling. Cyclobutylidene has a nonclassical bonding structure and is stable enough to be captured in bimolecular reactions. Cyclobutanediazirines have more recently been studied as photoaffinity probes based on cyclobutylidene, but the mechanism, especially with respect to the role of putative diazo intermediates, was not fully understood. Here, we show that photolysis (365 nm) of cyclobutanediazirines can produce cyclobutylidene intermediates as evidenced by formation of their expected bimolecular and unimolecular products, including methylenecyclopropane derivatives. Unlike linear diazirines, cyclobutanediazirine photolysis in the presence of tetramethylethylene produces a [2+1] cycloaddition adduct. By contrast, linear diazirines produce diazo compounds upon low temperature photolysis in THF, whereas diazo compounds are not detected in similar photolyses of cyclobutanediazirines. Diazocyclobutane, prepared by independent synthesis, is labile, reactive toward water, and capable of protein alkylation. The rate of diazocyclobutane decomposition is not affected by 365 nm light, suggesting that the photochemical conversion of diazocyclobutane to cyclobutylidene is not an important pathway. Finally, chemical proteomic studies revealed that a likely consequence of this primary conversion to a highly reactive carbene is a marked decrease in labeling by cyclobutanediazirine-based probes relative to linear diazirine counterparts both at the individual protein and proteome-wide levels. Collectively, these observations are consistent with a mechanistic picture for cyclobutanediazirine photolysis that involves carbene chemistry with minimal formation of diazo intermediates, and contrasts with the photolyses of linear diazirines where alkylation by diazo intermediates plays a more significant role.

Project description:PRIDE > Archive > PXD013420 Project PXD013420 Download Project Files Summary Title A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Description Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site hotspot maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling of the ternary complex based on the binding site map revealed a 5.0 Å minimum distance constraint between the conjugated residues and the diazirine carbon. Molecular dynamics further predicted a 9.0 Å labeling radius for the diazirine upon photo-activation that may be useful in the interpretation of binding site measurements from PAL more broadly. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted a precise structural measurement for interpretation of PAL products that may enable the discovery of new ligand space in the “undruggable” proteome.

Dataset Information

Deciphering Diazirine Reaction Mechanism: through Irradiation Modulation for Residue-Specific Distance Restraints in Protein Photo-Crosslinking

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets