Probing the Tertiary Structure of Multidomain Proteins by Limited Proteolysis and Mass Spectrometry

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) 1–130), the still covalently linked output-and C-terminal domains (OC; aa 133–469), the C-terminal domain (C; aa 397–469), 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 NtrC_R, 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 NtrC_R in the A4-helix and the B4-β-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.