Poster abstracts

Poster number 20 submitted by Pepsi Holmquist

Investigating the disassembly pathway of the Anti-TRAP dodecameric complex by surface induced dissociation mass spectrometry (SID-MS)

Melody Pepsi Holmquist (OSBP), Sophie Harvey, Vicki Wysocki, Mark Foster (Department of Chemistry and Biochemistry, OSU)

Abstract:
In bacillus, production of the enzymes responsible for biosynthesis of the essential amino acid tryptophan (Trp) is tightly regulated through a complex feedback loop involving two oligomeric proteins, one of which also binds tryptophan and RNA. When there is abundant Trp in the cell, transcription of the genes responsible for its biosynthesis, encoded in the trp operon, is inhibited by the trp RNA-binding attenuation protein (TRAP). Trp-activated TRAP binds to the 5’ leader region of the trp mRNA, resulting in both transcription attenuation and translation inhibition. To balance the repression by TRAP, another protein, Anti-TRAP (AT) binds TRAP, inhibiting RNA binding and thereby allowing the Trp biosynthesis genes to be expressed. AT is an oligomeric protein that exists in a pH-dependent equilibrium between a trimer AT3 and a dodecamer AT12, while only the trimeric form can bind TRAP. AT12 consists of a tetrahedral arrangement of four AT3, whose mechanism of assembly and disassembly represents a fascinating puzzle. To understand how inactive AT12 dissociates into active AT3 we are using surface-induced dissociation mass spectrometry (SID-MS). In SID-MS, selected ions known as the complex are dissociated via collisions with a fluorocarbon coated gold surface, and the resulting ions are then detected. The SID-MS approach is similar to that of CID (collision induced dissociation), in which ions are observed after multiple collisions with an inert gas, but fragmentation results from a single collision with the surface, which can result in less unfolding and more native structures for the dissociation products. Because formation of AT12 is dependent on pH and proper deformylation of the protein’s N-terminus, we will illustrate how these and other variables influence dissociation of inactive AT 12-mers to form trimers that are able to bind and reverse the inhibitory effect of TRAP on tryptophan biosynthesis.

Keywords: mass spectrometry, Anti-TRAP