Dataset Information

Design and evaluation of a cyclobutane diazirine alkyne tag for photoaffinity labeling in cells

ABSTRACT: Alkyl diazirines are frequently used in photoaffinity labeling to trap and identify small molecule–protein interactions. How-ever, the alkyl diazirine can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a long-lived alkyl diazo intermediate. To alter this reactivity preference, we developed PALBOX, a novel cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity. We show that PALBOX is readily derivatized and attached to small molecules to profile their binding interactions in cells. Systematic experiments with biological sub-strates show that the cyclobutane diazirine scaffold does not exhibit reactivity characteristic of unconstrained alkyl diazo in-termediates. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label known protein targets in cells with higher specificity. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule–protein binding sites. Thus, PALBOX is a versatile diazirine-based pho-toaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule–protein interactions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christina Woo

LAB HEAD: Christina M. Woo

PROVIDER: PXD035954 | Pride | 2024-01-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	211021LMRSAM09417_YA-III-154_LENBOX.msf	Msf
	211021LMRSAM09417_YA-III-154_LENBOX.raw	Raw
	211112L_SAM0956.msf	Msf
	211112L_SAM09566_AW_VI_100_Fr1.raw	Raw
	211112L_SAM09567_AW_VI_100_Fr2.raw	Raw

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Publications

Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.

West Alexander V AV Amako Yuka Y Woo Christina M CM

Journal of the American Chemical Society 20221109 46

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reac ...[more]

PMID: 36350779

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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.