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Crosslinking mass spectrometry with novel aryl‐dialdehyde reagents reveal extended conformational dynamics of RNA polymerase II
Author(s) -
Trnka Michael J,
Burlingame A. L.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.797.4
Subject(s) - chemistry , reagent , dissociation (chemistry) , mass spectrometry , electron transfer dissociation , bifunctional , aryl , combinatorial chemistry , polymerase , chromatography , biochemistry , tandem mass spectrometry , organic chemistry , dna , catalysis , alkyl
Chemical crosslinking mass spectrometry is a means of probing the structures of protein machines in their native states. Bifunctional reagents covalently label stable complexes, reflecting their topology. Identification of the modified sites generates distance restraints, which together with crystallography of individual subunits and cryoEM visualization of entire complexes, can be used to model large three‐dimensional structures. Furthermore, solid‐state protein structures are conformationally constrained but crosslinking can reveal the extent of their dynamics. Here, we probe RNA polymerase II (pol II) with aryl‐dialdehyde (ADA) crosslinking reagents. ADA reagents preserve positive charge at the site of crosslinking, which facilitates the enrichment of crosslinked peptides by cation exchange chromatography relative to conventional reagents. Furthermore, these highly charged precursors are well suited to analysis by electron transfer dissociation (ETD) mass spectrometry, which provides more comprehensive product ion coverage than collisional dissociation and facilitates identification of crosslinked peptides in complex searches. The data indicate that the pol II clamp domain is more dynamic than indicated by crystallography. Funding was provided by the Biomedical Technology Research Centers program of the NIH National Institute of General Medical Sciences NIH NIGMS 8P41GM103481