Essentially all cellular processes are regulated and driven by certain protein-protein interactions (PPIs) and dysfunction is related to various diseases. Especially photo-crosslinking represents a powerful tool to study these crucial noncovalent interactions. Different photoreactive groups are described in literature such as benzophenones and diazirines with the latter ones being known for their small size and high reactivity. These two properties ensure high cross-linking specificity and efficiency upon photoactivation, as diazirines can be installed close enough to the desired reaction site without inducing undesired steric hindrance.

Thus, photoaffinity labeling (PAL) with diazirines is a convenient method to identify noncovalent interactions of biomolecules. Depending on the cellular environment, labeling requirements can vary, hence, a set of different diazirine labeling agents is required to find the best conditions. 

Amines, carboxylic acids, and in particular amino acids decorated with a diazirine moiety exhibit preferential labeling in a pH-dependent manner either via a diazo or a carbene intermediate. If electronegative atoms are in close proximity, e.g. as particularly in the case of aryl-fluorodiazirines, the carbene pathway is preferred. With this toolbox, alkyl diazirine probes can be used to preferentially label highly acidic proteins or those embedded in membranes. Probes carrying a positive charge will produce higher labeling yields in cells and in vitro.

Protein-protein interactions (PPI) within a pH neutral environment (left) and an electrostatically charged environment.

Diazirine-substituted fatty acid cross-linkers bearing an additional functional group for click chemistry (azide or alkyne) can be used for the detection of membrane and transmembrane phenomena. The long alky chain acts as anchor within the membrane’s lipid chain, while the carboxylic acid function can be conjugated to nucleophilic functions of the substrate of interest. The diazirine ring will dismiss dinitrogen upon irradiation with UV light and the remaining carbene will undergo a spontaneous insertion addition reaction with any neighboring molecule forming a covalent bond.

Trifunctional fatty acid derivatives enable sophisticated membrane studies. The acid residue can be used for conjugation to any substrate or probe for detection. The diazirine allows covalent linkage to the protein of interaction upon irradiation with UV light and the alkyne function still offers additional options, like attachment of Biotin-azide for isolation of the complex.

→  For more information about photo-crosslinkage in living cells with diazirine amino acids check out our blog! 

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References:

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A Genetically Encoded Diazirine Analogue for RNA-Protein Photo-crosslinking; D. Dziuba, J. E. Hoffmann, M. W. Hentze, C. Schultz; Chembiochem : a European journal of chemical biology 2020; 21: 88-93. https://doi.org/10.1002/cbic.201900559

A bifunctional amino acid to study protein-protein interactions; T. Yang, X. Li, X. D. Li; RSC Adv 2020; 10: 42076-42083. https://doi.org/10.1039/d0ra09110c

Global Mapping of Protein-Lipid Interactions by Using Modified Choline-Containing Phospholipids Metabolically Synthesized in Live Cells; D. Wang, S. Du, A. Cazenave-Gassiot, J. Ge, J.-S. Lee, M. R. Wenk, S. Q. Yao; Angew. Chem. Int. Ed. Engl. 2017; 56(21): 5829-5833. https://doi.org/10.1002/anie.201702509

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells; D. Höglinger, A. Nadler, P. Haberkant, J. Kirkpatrick, M. Schifferer, F. Stein, S. Hauke, F. D. Porter, C. Schultz; PNAS 2017; 114(7): 1566-1571. https://doi.org/10.1073/pnas.1611096114

Exploring the Binding Proteins of Glycolipids with Bifunctional Chemical Probes; X. Liiu, T. Dong, Y. Zhou, N. Huang, X. Lei; Angew. Chem. Int. Ed. 2016; 55(46): 14330-14334. https://doi.org/10.1002/anie.201608827

Bifunctional Fatty Acid Chemical Reporter for Analyzing S-Palmitoylated Membrane Protein-Protein Interactions in Mammalian Cells; T. Peng, H. C. Hang; J. Am. Chem. Soc. 2015; 137(2): 556-559. https://doi.org/10.1021/ja502109n

In Vivo Profiling and Visualization of Cellular Protein-Lipid Interactions Using Bifunctional Fatty Acids; P. Haberkant, R. Raijmakers, M. Wildwater, T. Sachsenheimer, B. Brügger, K. Maeda, M. Houweling, A.-C. Gavin, C. Schultz, G. van Meer, A. J. R. Heck, J. C. M. Holthuis; Angew. Chem. Int. Ed. 2013; 52(14): 4033-4038. https://doi.org/10.1002/anie.201210178

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Cross-linking chemistry and biology: development of multifunctional photoaffinity probes. T. Tomohiro, M. Hashimoto, Y. Hatanaka; Chem. Record 2005; 5(6): 385-395. https://doi.org/10.1002/tcr.20058