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Using FlAsH To Probe Conformational Changes in a Large HEAT Repeat Protein
Author(s) -
Tsytlonok Maksym,
Itzhaki Laura S.
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201200012
Subject(s) - biophysics , protein folding , chemistry , structural motif , fluorescence , affinities , biochemistry , biology , physics , quantum mechanics
We have investigated the use of FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, to probe folding of the 15‐HEAT repeat protein PR65A. PR65A is one of a special class of modular non‐globular proteins known as tandem repeat proteins, which are composed of small structural motifs that stack to form elongated, one‐dimensional architectures. We were able to introduce linear and bipartite tetracysteine motifs at several sites along the α‐helical HEAT array of PR65A without disrupting the structure or stability. When the linear tetracysteine motif CCPGCC was used, FlAsH fluorescence reported globally on the folding of the protein. When the tetracysteine motif was displayed in bipartite mode through the engineering of pairs of cysteines on adjacent HEAT repeats, FlAsH fluorescence became a reporter of local conformation and of oligomerisation. Thus, by designing FlAsH‐binding sites at different locations along the repeat array one can interrogate specific properties of PR65A, paving the way for structure–function analysis of this protein both in vitro and in the cell.