Chemical crosslinking and LC/MS analysis to determine protein domain orientation: Application to AbrB

• Published in: Biochemical and biophysical research communications Vol. 431; no. 2; pp. 253 - 257
• Main Authors: Olson, Andrew L., Liu, Fan, Tucker, Ashley T., Goshe, Michael B., Cavanagh, John
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.02.2013
• Subjects: AbrB; Amino Acid Sequence; Bacterial Proteins - chemistry; Chemical crosslinking; Chromatography, Liquid; Cross-Linking Reagents - chemistry; DNA-Binding Proteins - chemistry; Domain orientation; Gas-phase cleavable; Molecular Sequence Data; NMR; Protein Structure, Tertiary; Solution structure; Tandem Mass Spectrometry; Transcription Factors - chemistry; PRE; NMR; Solution structure; NOE; LC/MSn; Domain orientation; SuDP; FRET; Chemical crosslinking; AbrB; Gas-phase cleavable
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2012.12.124

► The transition state regulator AbrB from Bacillus subtilis exists as a functional tetramer. ► Inherent dynamics makes AbrB extremely challenging to investigate from conventional methods. ► Chemical crosslinking and MS analysis to generate a model of full length AbrB from B. subtilis. To fully understand the modes of action of multi-protein complexes, it is essential to determine their overall global architecture and the specific relationships between domains and subunits. The transcription factor AbrB is a functional homotetramer consisting of two domains per monomer. Obtaining the high-resolution structure of tetrameric AbrB has been extremely challenging due to the independent character of these domains. To facilitate the structure determination process, we solved the NMR structures of both domains independently and utilized gas-phase cleavable chemical crosslinking and LC/MSn analysis to correctly position the domains within the full tetrameric AbrB protein structure.