Soft-ionisation methods, particularly e!ectrospray (ESI) and matrix-assisted laser desorption (MALDI) have enabled a breakthrough in the mass spectrometric structure analysis of proteins. Whereas ESI-MS provides direct information about solution structures and non-covalent interactions, the combination of mass spectrometry with structure-specific protein chemistry is emerging presently as a powerful tool for characterising tertiary structures and structure-function relations. Recent developments of chemical modification reactions are summarised which are suitable to the mass spectrometric analysis of reactive sites, surface topology and antigenic determinants in protein-tertiary structures, and can be efficiently employed in x-ray crystallographic structure determinations. Applications to the structure elucidation, and functional characterisation of porin-channel proteins and to leucine zipper protein-nucleotide complexes illustrate their efficiency in the analysis of two most important topics of structural biology, molecular recognition structures and biomacromolecular interaction.
Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions.
The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel -strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant ␣-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(⌬1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.Keywords: ScrY porin; N-terminal periplasmic domain; coiled-coil; circular dichroism; mass spectrometry; TR-NOE-NMR spectroscopy; supramolecular association.A sucrose-specific uptake channel, originally discovered by Schmid et al. (1982), has been found on plasmid pUR400 in Salmonella typhimurium and Escherichia coli that confers to the cells, under growth conditions, the ability to grow on sucrose as a sole carbon source. The structure of the sucrose-specific porin (ScrY) from S. typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel -strands ( Fig. 1; Forst et al. 1998). However, the 71-residue N-terminal periplasmic polypeptide sequence of ScrY porin could not be traced and modeled in the crystal structure (Schmid et al. 1982;Forst et al. 1998) due to the absence of electron density. Mature ScrY porin consists of 483 residues. A comparison of the amino acid sequence of ScrY porin with that of maltoporin (LamB) revealed a re-
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.