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Eur. J. Mass Spectrom. 4, 279 - 285 (1998) |
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Probing the tertiary structure of multidomain proteins by limited proteolysis and mass spectrometry | ||
Marcus Bantscheff and Michael O. Glocker* |
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ABSTRACT: | ||
Limited proteolysis of the multidomain nitrogen regulatory protein C (NtrC) with thermolysin revealed well separated fragments using high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) molecular weight determinations from the fragment mixtures showed that the cleavage products resembled the N-terminal receiver domain (R; amino acids (aa) 1130), the still covalently linked output- and C-terminal domains (OC; aa 133469), the C-terminal domain (C; aa 397469), a core-fragment of the O-domain (O*), and intact NtrC. Borders of the separated domains were identified by mass spectrometric peptide mapping after on-target proteolysis of the HPLC-separated fragments. The flexible and, hence, accessible linker region between the R- and the O-domains of NtrC was shown to comprise the amino acids Val-131 and Gln-132. Thermolysin split the OC-fragment into the O- and the C-domains at accessible amino acid residues ranging from Thr-389 to Gln-396 identifying this partial sequence as a second hitherto unknown linker in NtrC. Individually expressed NtrCR, the R-domain of NtrC, afforded structure-dependent proteolytic fragments on tryptic digestion in solution. Mass spectrometric peptide mapping analyses determined the locations of cleavages in NtrCR in the A4-helix and the B4-b-sheet/loop region, providing information on surface-exposed partial structures of the R-domain. The combination of limited proteolysis with micro-preparative techniques and mass spectrometry provides an efficient tool for the rapid identification of protein tertiary structural features, affording lead information necessary for protein design and engineering and for structure/function studies. | ||
Keywords: Two-component signal transduction proteins, on-target proteolysis, structure/function correlation, MALDI-MS, ESI-MS. |
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