Identification of Linker Regions and Domain Borders of the Transcription Activator Protein NtrC from Escherichia coli by Limited Proteolysis, In-Gel Digestion, and Mass Spectrometry
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- 作者：Marcus Bantscheff ; Verena Weiss ; and Michael O. Glocker
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 24, 1999
- 年：1999
- 卷：38
- 期：34
- 页码：11012 - 11020
- 全文大小：137K
- 年卷期：v.38,no.34(August 24, 1999)
- ISSN：1520-4995
文摘
We have developed a mass spectrometry based method for the identification of linker regionsand domain borders in multidomain proteins. This approach combines limited proteolysis and in-gelproteolytic digestions and was applied to the determination of linkers in the transcription factor NtrCfrom Escherichia coli. Limited proteolysis of NtrC with thermolysin and papain revealed that initialdigestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domainand the still covalently linked OC-domains. Subsequent steps in limited proteolysis afforded further cleavageof the OC-fragment into the O- and the C-domain at accessible amino acid residues. Mass spectrometricidentification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker betweenR- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement withpapain and subtilisin digestion. The region between amino acid residues Thr-389 and Gln-396 markedthe hitherto unknown linker sequence that connects the O- with the C-domain. High abundances of proline-,alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of relatedNtrC response regulator proteins. While R- and C-domains remained stable under the applied limitedproteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised thesequence from Ile-140 to Arg-320. ATPase activity was lost after separation of the R-domain from theOC-fragment. However, binding of OC- and C- fragments to specific DNA was observed by characteristicband-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain. The outlinedstrategy proved to be highly efficient and afforded lead information of tertiary structural features necessaryfor protein design and engineering and for structure-function studies.