X-Ray Crystallographic and Mass Spectrometric Structure Determination and Functional Characterisation 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 lysinet-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal a-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.

Single-Walled Carbon Nanotubes: Mimics of Biological Ion Channels

“…1 , 27 , 28 In comparison, native (with no negative charges at the pore entrance) and succinylated porins ( Rhodobacter capsulatus , negatively charged pore) and Hemolysins (negatively charged pore) display P c / P a in KCl of ∼9, ∼ 23, and ∼9, respectively. 25 , 29 Similarly, the selectivity of cation/anion in leakage or control devices (without a SWCNT) is poor and displays P c / P a of ∼5 ( Figure S6 ).…”

Approaches to the Characterisation of Tertiary and Supramolecular Protein Structures by Combination of Protein Chemistry and Mass Spectrometry

“…Generally, they form trimeric complexes consisting of 16-18-stranded ~-barrels of high stability, which have a similar periplasmic pore mouth but different loops in the medium-opposed mouths (s. Figures 7 and 8, and ref. [43], this volume). The structures, transport functions and selectivities, and membrane association of general diffusion channels as well as specific porins have been subject of detailed studies by several groups in the last years, both in the soluble and the solid (crystalline) state [25,43,50].…”

“…[43], this volume). The structures, transport functions and selectivities, and membrane association of general diffusion channels as well as specific porins have been subject of detailed studies by several groups in the last years, both in the soluble and the solid (crystalline) state [25,43,50]. Methods for the direct mass spectrometric characterisation of porins in solubilised, detergent-containing preparations have been recently developed [51][52][53].…”

“…A characteristic structure element of Rc.-porin is a central constriction loop inside the p-barrel cylinder which constricts the free cross section to approximately 8x lOA and shows a highly asymmetric charge distribution of acidic and basic amino acid~; opposite to each other [51]. Ion conductance studies with lipid membranes in vitro have strongly suggested this peptide loop as the central site determining cation/anion permeability and transport selectivity [43]. The functional role of charges near the pore mouth of Rc.-porin and other ion channels has been intensively discussed in theoretical work.…”

“…at a 1.4 A van der Waals sphere of solvent accessibility [41)) for several proteins with known structures, whereas pK.-differences were only found significant for the rapid N-terminal acylation [17,42]. This feature has provided useful molecular information on surface topology and accessible reactive sites, as illustrated in structure-function studies of porin ion-channel proteins (see also ref [43], this volume). In hbn egg-white lysozym (HEL), the most reactive Lys-97 residue has the highest surface accessibility [17].…”