The ability to label specific proteins using photoactivatable small molecule probes with inherent affinity for explicit protein substrates, or photolabeling, has a vast array of applications.  Examples range from target identification of bioactive small molecules to development of selective inhibitors for a particular protein. While these applications may be diverse in nature, they all require the ability for these probes to specifically crosslink to their targets and necessitate reliable detection of the resulting covalent protein-probe adduct.  The aim of this research was to improve upon previous photolabeling studies of a group of hydroxamate-based photoreactive inhibitors/probes of histone deacetylases (HDACs) in HeLa cell lysates.To improve upon the specificity and efficiency of photocrosslinking, we compared probes that used an aryl azide (ArN3) to probes that used a tetrafluoro aryl azide (4FArN3) for photoactivation and covalent addition to histone deacetylase.  To improve upon the detection we compared probes that contained an alkyl azide substituent used to react with an alkyne conjugated tag, to probes that contained an alkyne substituent used to react with an azide conjugated tag. Several different tags were also explored: alkyne or azide conjugated biotin, IR dye, and His6. We demonstrated that differences in the specificity and efficiency of covalent crosslinking of the ArN3 probes and the 4FArN3 probes to HDACs could be distinguished using SDS-PAGE coupled with fluorescent detection, and that these differences were affected by the wavelength of irradiation used to activate the probes.  Furthermore, we found that incorporating an alkyne substituent on the probe to react with an azide detection tag decreased the amount of nonspecific photolabeling of proteins by the probes.  Finally, we recognized that using an azide or alkyne conjugated biotin tag to react with the probes to be recognized by a streptavidin conjugated fluorophore can obscure interpretation of labeled proteins from cell lysates.

Optimization of photolabeling and visualization of histone deacetylase 2 in cell lysates

248th National Meeting (2014)

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