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Anal Chem. 2009 Mar 15;81(6):2218-26. doi: 10.1021/ac802354j.

RNA and protein complexes of trp RNA-binding attenuation protein characterized by mass spectrometry.

Author information

1
Yokohama City University, Supramolecular Biology, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. akashi@tsurumi.yokohama-cu.ac.jp

Abstract

We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution.

PMID:
19219981
DOI:
10.1021/ac802354j
[Indexed for MEDLINE]

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