Volume 2g0, number 2, 379~3~ FEELS 09~)4 '~ 1991 Feder#tton of I~aroF~an ltio,~heml¢~l So~iett,¢i 0014~17~1~'91t$] Tile structure of the perth front Rlmd.b~trt¢r ¢cJpxlqhuu~ was determin¢d at a resolution of !,~ A. The analysi=t stalled from a eJt~sety r~laled ¢rytl¢il ~tructur~ thai had been sol~'ed at a medium resolution of 3 A using mulliple isotnorphou~ replacement ttnd solvent llattenin//,. The new structure contains the complete seq uent:e of.~0 ! amino acid residues, Refinement of the model is uoder ~¢~y; the present R.fa¢ior i~ 22,~ with ~ood geometry,Except for the len~lths of several loops, the resultinl~ chain fold corresponds to the medium r0soltttion model, The membrane cha,mel is lined by a larl/e naml:er of ionol|cnic side chains with ¢:haracttriliie segrel~ation ofdifl'~rently charged ~troups.

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.

No abstract

The primary structure of the integral membrane protein porin from the purple bacterium Rhodobacter capsulatus was determined. The protein was cleaved with trypsin, CNBr and Asp-N protease. The peptides were isolated, sequenced and aligned to a total length of 301 residues with an M , of 31 536. The low isoelectric point of 3.9 is confirmed by the high excess of 34 Asp and 17 Glu (16.9%) over 10 Lys, 7 Arg and 2 His (6.3%). Overall sequence similarity to other porins is not evident when using sequence alignment programs. However, a partial relationship to Neisseriu porins seems to exist. The established sequence has been used as the basis for a threedimensional structure determination by X-ray diffraction at 0.18-nm resolution. The arrangement of the sequence in the 16-stranded P-barrel of porin is given. Some sequence-structure correlations are discussed.Porins are pore-forming proteins in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts. The channels allow for the passive diffusion of small hydrophilic solutes up to an exclusion limit of about 0.6 kDa [l -31. In general, porins form trimers that are relatively stable towards high temperature, proteases and detergents. The primary structure of more than 40 porins from both bacteria and eukaryotes including man have been elucidated. Some of them are clearly homologous with each other. Homologies are observed between porins from different species, and porins of different specificities within one species like OmpC, OmpF and PhoE in Escherichia coli. However, most groups of homologous porins can hardly be related to each other on the basis of their sequences alone. This is also reflected in the sizes of the known porin sequences which range from 282-483 residues/monomer.The general structure of porin from the phototrophic bacterium Rhodobacter capsulatus was determined by X-ray diffraction analysis [4]. It showed that the trimer consists of three 16-stranded fl-barrels forming three pores that merge on one side. The sequence of this porin presented here has been used for a detailed interpretation of the electron density map derived from X-ray diffraction yielding the complete structure. After a recent improvement of the resolution to 0.18 nm [5-71, the structure is now known in exact atomic detail, showing the shape and the inner lining of the channels as well as the interfaces between the monomers and the protein surface contacting the membrane. MATERIALS AND METHODS MaterialsTrypsin and the Asp-N and Glu-C proteases were sequencing grade and carboxypeptidase Y normal grade from Boehringer-Mannheim. Chymotrypsin treated with tosyllysylchloromethane was purchased from Sigma. Sequencer reagents were from Applied Biosystems or Fluka. Q-Sepharose fast flow and Sephacryl S-200 superfine were obtained from Pharmacia. Other chemicals were of analytical grade. Isolation of porinPorin was isolated as the trimer from R. capsulatus strain 37b4 [5, 71 using a modification of the procedures of [8 -101. 25 g thawed cells were washed with 10 mM MgC12, 2...

The crystal electron density map of porin from Rhodobacter capsulufus 37b4 at 0.6 nm resolution shows that the trimeric molecule consists of 3 merged cylinders as the central part, plus 3 laterally radiating domains. The density shows no prominent a-helices and is consistent with p-pleated sheet structure. The trimer density was dissected into monomers. Three separate pores per trimer with sizes that agree with the exclusion limit of permeating molecules could be identified. The cross-section of the central part as well as the pore distance agree with prior electron microscopy data on other porins.Porin; Membrane protein structure; X-ray structure; (Rhodobacter capsulutus)