X-RAY CRYSTALLOGRAPHIC AND MASS-SPECTROMETRIC STRUCTURE DETERMINATIONAND FUNCTIONAL-CHARACTERIZATION OF SUCCINYLATED PORIN FROM RHODOBACTER-CAPSULATUS - IMPLICATIONS FOR ION SELECTIVITY AND SINGLE-CHANNEL CONDUCTANCE

The role of charges near the pore mouth has been discussed in theoreti cal work about ion channels. To introduce new negative charges in a ch annel 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 determine d by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ioniz ation mass spectrometry identified selective succinylation of three ly sine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-termina l alpha-amino group. The structure of a tetra-succinylated porin (TS-p orin) was determined to 2.4 alpha and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylat ed 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-a ccessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic ent rance of the channel. The Lys-46 residue at the central constriction l oop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical proper ties of the MS- and TS-porins were determined by reconstitution into b lack lipid membranes, and showed a negative charge effect on ion trans port and an increased cation selectivity through the porin channel. Th e 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 aqueou s salt concentration. The substantially higher cation selectivity of t he succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These resul ts show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion c hannels, and X-ray crystallography and mass spectrometry as complement ary analytical tools for defining precisely the chemically modified st ructures.