Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.     

Folding patterns of porin and bacteriorhodopsin.

Porin spans the outer membrane of Escherichia coli with most of the protein embedded within the membrane. It lacks pronounced hydrophobic domains and consists predominantly of beta-pleated sheet. These observations require the accommodation of polar and ionizable residues in an environment that has a low dielectric constant. Owing to a currently limited understanding of the constraints governing membrane protein structure, a minimal approach to structure prediction is proposed that identifies segments causing polypeptides to reverse their direction (turn identification). The application of this procedure avoids hydrophobicity parameters and yields a model of porin which is in good agreement with all experimental data available. The presence of polar and ionizable residues within membrane boundaries implies a dense (saturating) network of hydrogen bond donor and acceptor groups. Application to a paradigm of hydrophobic membrane proteins, bacteriorhodopsin, reveals a pattern consistent with its alpha-helical folding. The postulated structure includes significantly more polar residues in the membrane domain than have been assumed previously, suggesting that there are also hydrogen bonding networks in bacteriorhodopsin. Extensive networks permeating protein interior and surfaces would explain the extraordinary stability and the tight interactions between functional units in the formation of crystalline arrays of both proteins.     

Trigonal crystals of porin from Escherichia coli

Trigonal crystals of the integral membrane protein porin from Escherichia coli have been grown and characterized. They belong to space group P321 with unit cell constants a = b = LL8.4, c = 52.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals grow as well-defined hexagonal prisms to a size of 0.25 mm in all dimensions, and diffract to 2.7 A. The molecular symmetry coincides with 3-fold crystallographic symmetry, giving two trimers per unit cell (1 monomer/asymmetric unit). This corresponds to VM = 2.9 A3/Da. Native X-ray data to 3.0 A resolution have been collected on a FAST area detector and a search for heavy atom derivatives is underway.     

Three-dimensional crystals of an integral membrane protein: an initial x-ray analysis

Matrix protein, a pore-forming transmembrane protein spanning the outer membrane of Escherichia coli, has been obtained in a variety of three- dimensional crystal forms amenable to both electron microscope and x- ray analyses. Successful association into large crystals depended on the use of alpha-octyl glucoside, a detergent with relatively low affinity for the protein. Electron micrographs of thin-sectioned crystals show a high degree of order. Preliminary crystallographic data suggest that the crystals, which exhibit diffraction to 3.8 A, have a cubic space group.     

Stability of membrane proteins: relevance for the selection of appropriate methods for high-resolution structure determinations

High stability is a prominent characteristic of integral membrane proteins of known atomic structure. But rather than being an intrinsic property, it may be due to a selection exerted by biochemical procedures prior to structure determination, since solubilization results in the transient exposure of membrane proteins to solution conditions. This may cause structural perturbations that interfere with 3D crystallization and hence with X-ray analysis. This problem also affects the preparation of samples for electron crystallography and NMR studies and may account for the fact that high-resolution structures of representatives of whole groups, such as transport proteins and signal transducers, have not been elucidated so far by any method. A knowledge of the proportion of labile proteins among membrane proteins, and of the kinetics of their denaturation, is therefore necessary. Establishing stability profiles, developing methods to maintain lateral pressure, or preventing contact with water (or both) should prove significant in establishing the structures of conformationally flexible proteins.     

Identification and Characterization of Two Quiescent Porin Genes, nmpC and ompN, in Escherichia coli BE

The genomic DNA of the B^E strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli B^E.