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Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
Published on: December 17, 2013
Analysis of protein-protein interaction of the mitochondrial translocase at work by using technically effective BPA
Edward Germany1, Takuya Shiota1
1Frontier Science Research Center, University of Miyazaki, Kiyotake, Miyazaki, Japan.
Abstract:
The BPA photo-crosslinking method exploits the property of p-benzoyl-L-phenylalanine (pBpa), an amino acid containing a photoreactive side chain, and allows for the crosslinking with nearby proteins upon Ultraviolet irradiation. This feature enables the capture of two proteins within a close proximity with high spatial resolution at the level of amino acid residues. In this chapter, we introduce an example of the employment of the BPA photo-crosslinking method to the Translocase of the Outer Mitochondrial membrane complex of mitochondria in Saccharomyces cerevisiae as a model protein translocase. Here in, we provide three procedures (i) the introduction of pBpa into proteins of interest in living yeast cells by in vivo suppressor tRNA system; (ii) analysis of in vivo subunit-subunit interactions intra-complex; and (iii) analysis of translocase channel-substrate interactions in organello. The use of in vivo and in organello crosslinking tools enable the robust analysis of translocases in a near-to physiological condition.
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