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 m ass spectrometry (MALDIMS) and electrospray ionization mass spectrometry (E SI-MS) molecular weight determinations from the fragment mixtures showed th at the cleavage products resembled the N-terminal receiver domain (R; amino acids (aa) 1-130), the still covalently linked output- and C-terminal doma ins (OC; aa 133-469), the C-terminal domain (C; aa 397-469), a core-fragmen t of the O-domain (O*), and intact NtrC. Borders of the separated domains w ere identified by mass spectrometric peptide mapping after on-target proteo lysis of the HPLC-separated fragments, The flexible and, hence, accessible linker region between the R- and the O-domains of NtrC was shown to compris e the amino acids Val-131 and Gln-132, Thermolysin split the OC-fragment in to 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 u nknown linker in NtrC, Individually expressed NtrC(R), the R-domain of NtrC , afforded structure-dependent proteolytic fragments on tryptic digestion i n solution. Mass spectrometric peptide mapping analyses determined the loca tions of cleavages in NtrC(R) in the A4-helix and the B4-beta-sheet/loop re gion, providing information on surface-exposed partial structures of the R- domain, The combination of limited proteolysis with micro-preparative techn iques and mass spectrometry provides an efficient tool for the rapid identi fication of protein tertiary structural features, affording lead informatio n necessary for protein design and engineering and for structure/function s tudies.