title: X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.
abstract: The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.
authors.affiliation: Universitat Konstanz, Fakultat fur Chemie, Germany. michael.przybylski@uni-konstanz.de
authors.forename: M
authors.initials: M
authors.lastname: Przybylski
authors.forename: M O
authors.initials: MO
authors.lastname: Glocker
authors.forename: U
authors.initials: U
authors.lastname: Nestel
authors.forename: V
authors.initials: V
authors.lastname: Schnaible
authors.forename: M
authors.initials: M
authors.lastname: Blüggel
authors.forename: K
authors.initials: K
authors.lastname: Diederichs
authors.forename: J
authors.initials: J
authors.lastname: Weckesser
authors.forename: M
authors.initials: M
authors.lastname: Schad
authors.forename: A
authors.initials: A
authors.lastname: Schmid
authors.forename: W
authors.initials: W
authors.lastname: Welte
authors.forename: R
authors.initials: R
authors.lastname: Benz
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003217:Conductometry; D018360:Crystallography, X-Ray; D003488:Cyanogen Bromide / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D011336:Probability; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016354:Rhodobacter capsulatus / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010713:Phosphatidylcholines; D018272:Porins; D014357:Trypsin; D008239:Lysine; D003488:Cyanogen Bromide