TMBHMM: a frequency profile based HMM for predicting the topology of transmembrane beta barrel proteins and the exposure status of transmembrane residues

Transmembrane beta barrel (TMB) proteins are found in the outer membranes of bacteria, mitochondria and chloroplasts. TMBs are involved in a variety of functions such as mediating flux of metabolites and active transport of siderophores, enzymes and structural proteins, and in the translocation across or insertion into membranes. We present here TMBHMM, a computational method based on a hidden Markov model for predicting the structural topology of putative TMBs from sequence. In addition to predicting transmembrane strands, TMBHMM also predicts the exposure status (i.e., exposed to the membrane or hidden in the protein structure) of the residues in the transmembrane region, which is a novel feature of the TMBHMM method. Furthermore, TMBHMM can also predict the membrane residues that are not part of beta barrel forming strands. The training of the TMBHMM was performed on a non-redundant data set of 19 TMBs. The self-consistency test yielded Q(2) accuracy of 0.87, Q(3) accuracy of 0.83, Matthews correlation coefficient of 0.74 and SOV for beta strand of 0.95. In this self-consistency test the method predicted 83% of transmembrane residues with correct exposure status. On an unseen, non-redundant test data set of 10 proteins, the 2-state and 3-state TMBHMM prediction accuracies are around 73% and 72%, respectively, and are comparable to other methods from the literature. The TMBHMM web server takes an amino acid sequence or a multiple sequence alignment as an input and predicts the exposure status and the structural topology as output. The TMBHMM web server is available under the tmbhmm tab at: http://service.bioinformatik.uni-saarland.de/tmx-site/.     

Prediction of secondary structure by evolutionary comparison: application to the alpha subunit of tryptophan synthase

The amino acid sequences of the a subunits of tryptophan synthase from ten different microorganisms were aligned by standard procedures. The alpha helices, beta strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived from profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted beta strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The a subunit is predicted to have eight repeated beta-loop-alpha-loop motifs with an extra N-terminal alpha helix and an intercalated segment of highly conserved residues. This pattern suggests that the territory structure of the a subunit is an eightfold alpha/beta barrel. The distribution of conserved amino acid residues and published data on limited proteolysis, chemical modification, and mutagenesis are consistent with the alpha/beta barrel structure. Both the active site of the a subunit and the combining site for the beta 2 subunit are at the end of the barrel formed by the carboxyl-termini of the beta strands.     

Structure and dynamics of the beta-barrel of the membrane transporter BtuB by site-directed spin labeling

Site-directed spin labeling and EPR spectroscopy were used to map two consecutive beta-strands of the putative transmembrane beta-barrel of BtuB. For these studies, a series of 29 consecutive single cysteine mutants of BtuB were produced covering residues 148-176. The proteins were then expressed, reacted with a sulfhydryl-specific spin label, purified in octyl glucoside (OG), and reconstituted into palmitoyloleoylphosphatidylcholine (POPC) bilayers. The labeled residues spanned from the extracellular region (position 148) to the small periplasmic loop (positions 160-163) and back up to the extracellular side (position 176) of BtuB. Continuous wave power saturation in the presence of oxygen or NiAA yielded an i, i + 2 periodicity for the collision frequencies at these sites and demonstrated the presence of a beta-strand structural motif. For both strands studied, the even-numbered residues were found to be exposed to the hydrophobic phase of the bilayer, whereas the odd-numbered residues pointed toward the interior of the barrel and the core of the protein. In addition, the collision parameters yielded the position of the protein within the bilayer. The phase relationship between the oxygen and metal collision frequencies along with the corresponding membrane depth parameters, Phi, indicates that segments 151-159 and 164-172 are within the bilayer. In POPC bilayers, there is a mobility gradient for spin labels along the barrel indicating enhanced backbone flexibility toward the periplasmic surface of the barrel. In POPC/OG mixed micelles, the even-numbered residues facing the hydrocarbon show an increased mobility compared with the bilayer environment whereas the inward-facing side chains show little change in motion. The data indicate that the protein core remains folded in POPC/OG mixed micelles but that this environment increases the backbone fluctuations of the strands. A model for the beta-barrel of BtuB is presented in part on the basis of these EPR data.     

Accurate prediction of the burial status of transmembrane residues of α-helix membrane protein by incorporating the structural and physicochemical features

Predicting the burial status (the residue exposure to the lipid bilayer or buried within the protein core) of transmembrane (TM) residues of α-helix membrane protein (αHMP) is of great importance for genome-wide annotation and for experimental researchers to elucidate diverse physiological processes. In this work, we developed a new computational model that can be used for predicting the burial status of TM residues of αHMP. By incorporating physicochemical scales and conservation index, an efficient prediction model using least squares support vector machine (LS-SVM) was developed. The model was developed from 43 protein chains and its prediction ability was evaluated by an independent test set of other non-redundant ten protein chains. The prediction accuracy of our method was much better than the results of the reported works. Our results demonstrate that the LS-SVM prediction model incorporating structural and physicochemical features derived from sequence information could greatly improve the prediction accuracy.     

Determining OMP topology by computation, surface plasmon resonance and cysteine labelling: the test case of OMPG

Bacterial outer-membrane proteins (OMP) are important in pathogenicity and the recently solved structure of OmpG provides an excellent test case for topological predictions since it is monomeric. Here we compare the results of applying several computerised structure prediction algorithms to the sequence of OmpG. Furthermore, we probe the OmpG topology by both an established chemical labelling approach and a new method which combines epitope insertion and surface plasmon resonance. The computational approaches are broadly accurate but the exact choice of the number of beta strands remains difficult. The algorithms also tend to predict the entire beta strand rather than just the transmembrane region. Epitope insertion clearly pinpoints exposed loops but its utility in defining buried or periplasmic sites is less clear cut. Cysteine-mutant labelling is largely confined to exposed residues but one periplasmic cysteine may be labelled by reagents entering via the OmpG pore.     

Topology prediction of Brucella abortus Omp2b and Omp2a porins after critical assessment of transmembrane beta strands prediction by several secondary structure prediction methods

In order to propose a reliable model for Brucella porin topology, several structure prediction methods were evaluated in their ability to predict porin topology. Four porins of known structure were selected as test-cases and their secondary structure delineated. The specificity and sensitivity of 11 methods were separately evaluated. Our critical assessment shows that some secondary structure prediction methods (PHD, Dsc, Sopma) originally designed to predict globular protein structure are useful on porin topology prediction. The overall best prediction is obtained by combining these three "generalist" methods with a transmembrane beta strand prediction technique. This "consensus" method was applied to Brucella porins Omp2b and Omp2a, sharing no sequence homology with any other porin. The predicted topology is a 16-stranded antiparallel beta barrel with Omp2a showing a higher number of negatively charged residue in the exposed loops than Omp2b. Experiments are in progress to validate the proposed topology and the functional hypotheses. The ability of the proposed consensus method to predict topology of complex outer membrane protein is briefly discussed.     

Structural predictions of AgfA, the insoluble fimbrial subunit of Salmonella thin aggregative fimbriae.

The unusually stable and multifunctional, thin aggregative fimbriae common to all Salmonella spp. are principally polymers of the fimbrin subunit, AgfA. AgfA of Salmonella enteritidis consists of two domains: a protease-sensitive, 22 amino acid residue N-terminal region and a protease-resistant, 109 residue C-terminal core. The unusual amino acid sequence of the AgfA core region comprises two-, five- and tenfold internal sequence homology patterns reflected in five conserved, 18-residue tandem repeats. These repeats have the consensus sequence, Sx5QxGx2NxAx3Q and are linked together by four or five residues, (x)xAx2. The predicted secondary structure for this unusual arrangement of tandem repeats in AgfA indicates mainly extended conformation with the beta strands linked by four to six residues. Candidate proteins of known structure with motifs of alternating beta strands and short loops were selected from folds described in SCOP as a source of coordinates for AgfA model construction. Three all-beta class motifs selected from the Serratia marcescens metalloprotease, myelin P2 protein or vitelline membrane outer protein I were used for initial AgfA homology build-up procedures ultimately resulting in three structural models; beta barrel, beta prism and parallel beta helix. The beta barrel model is a compact, albeit irregular structure, with the beta strands arranged in two antiparallel beta sheet faces. The beta prism model does not reflect the 5 or 10-fold symmetry of the AgfA primary sequence. However, the favored, parallel beta helix model is a compact coil of ten helically arranged beta strands forming two parallel beta sheet faces. This arrangement predicts a regular, potentially stable, C-terminal core region consistent with the observed tandem repeat sequences, protease-resistance and strong tendency of this fimbrin to oligomerize and aggregate. Positional conservation of amino acid residues in AgfA and the Escherichia coli AgfA homologue, CsgA, provides strong support for this model. The parallel beta helix model of AgfA offers an interesting solution to a multifunctional fimbrin molecular surface having solvent exposed areas, regions for major and minor subunit interactions as well as fiber-fiber interactions common to many bacterial fimbriae.     

Predicting transmembrane beta-barrels in proteomes

Very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence. One reason is that only very few high-resolution structures for transmembrane beta-barrel (TMB) proteins have been determined thus far. Here we introduced the design, statistics and results of a novel profile-based hidden Markov model for the prediction and discrimination of TMBs. The method carefully attempts to avoid over-fitting the sparse experimental data. While our model training and scoring procedures were very similar to a recently published work, the architecture and structure-based labelling were significantly different. In particular, we introduced a new definition of beta- hairpin motifs, explicit state modelling of transmembrane strands, and a log-odds whole-protein discrimination score. The resulting method reached an overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high as 86%. Furthermore, accurately discriminated TMB from non-TMB proteins (45% coverage at 100% accuracy). This high precision enabled the application to 72 entirely sequenced Gram-negative bacteria. We found over 164 previously uncharacterized TMB proteins at high confidence. Database searches did not implicate any of these proteins with membranes. We challenge that the vast majority of our 164 predictions will eventually be verified experimentally. All proteome predictions and the PROFtmb prediction method are available at http://www.rostlab.org/ services/PROFtmb/.     

Structural principles of alpha/beta barrel proteins: the packing of the interior of the sheet

Alpha/beta barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight-stranded beta-sheets and the packing of the residues at the center of the barrel. The packing in this region is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at some of the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues from the second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even-numbered strands that form the central layer, and side chains from the odd-numbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed beta barrel.     

Beta edge strands in protein structure prediction and aggregation

It is well established that recognition between exposed edges of beta-sheets is an important mode of protein-protein interaction and can have pathological consequences; for instance, it has been linked to the aggregation of proteins into a fibrillar structure, which is associated with a number of predominantly neurodegenerative disorders. A number of protective mechanisms have evolved in the edge strands of beta-sheets, preventing the aggregation and insolubility of most natural beta-sheet proteins. Such mechanisms are unfavorable in the interior of a beta-sheet. The problem of distinguishing edge strands from central strands based on sequence information alone is important in predicting residues and mutations likely to be involved in aggregation, and is also a first step in predicting folding topology. Here we report support vector machine (SVM) and decision tree methods developed to classify edge strands from central strands in a representative set of protein domains. Interestingly, rules generated by the decision tree method are in close agreement with our knowledge of protein structure and are potentially useful in a number of different biological applications. When trained on strands from proteins of known structure, using structure-based (Dictionary of Secondary Structure in Proteins) strand assignments, both methods achieved mean cross-validated, prediction accuracies of approximately 78%. These accuracies were reduced when strand assignments from secondary structure prediction were used. Further investigation of this effect revealed that it could be explained by a significant reduction in the accuracy of standard secondary structure prediction methods for edge strands, in comparison with central strands.     

A sequence-profile-based HMM for predicting and discriminating beta barrel membrane proteins

MOTIVATION: Membrane proteins are an abundant and functionally relevant subset of proteins that putatively include from about 15 up to 30% of the proteome of organisms fully sequenced. These estimates are mainly computed on the basis of sequence comparison and membrane protein prediction. It is therefore urgent to develop methods capable of selecting membrane proteins especially in the case of outer membrane proteins, barely taken into consideration when proteome wide analysis is performed. This will also help protein annotation when no homologous sequence is found in the database. Outer membrane proteins solved so far at atomic resolution interact with the external membrane of bacteria with a characteristic beta barrel structure comprising different even numbers of beta strands (beta barrel membrane proteins). In this they differ from the membrane proteins of the cytoplasmic membrane endowed with alpha helix bundles (all alpha membrane proteins) and need specialised predictors. RESULTS: We develop a HMM model, which can predict the topology of beta barrel membrane proteins using, as input, evolutionary information. The model is cyclic with 6 types of states: two for the beta strand transmembrane core, one for the beta strand cap on either side of the membrane, one for the inner loop, one for the outer loop and one for the globular domain state in the middle of each loop. The development of a specific input for HMM based on multiple sequence alignment is novel. The accuracy per residue of the model is 83% when a jack knife procedure is adopted. With a model optimisation method using a dynamic programming algorithm seven topological models out of the twelve proteins included in the testing set are also correctly predicted. When used as a discriminator, the model is rather selective. At a fixed probability value, it retains 84% of a non-redundant set comprising 145 sequences of well-annotated outer membrane proteins. Concomitantly, it correctly rejects 90% of a set of globular proteins including about 1200 chains with low sequence identity (<30%) and 90% of a set of all alpha membrane proteins, including 188 chains.     

Crystal structure of amylomaltase from thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans

Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the alpha-amylase superfamily of enzymes, whose core structure consists of a (beta, alpha)(8) barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the alpha-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the alpha-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the alpha-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the alpha-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.     

Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution

The crystal structure of recombinant human interleukin-1 beta (IL-1 beta) has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel beta-strands and no alpha-helix. The single 153-residue polypeptide chain is folded into a six-stranded beta-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the beta-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of beta-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1 beta receptors.     

NMR solution structure of the archaebacterial chromosomal protein MC1 reveals a new protein fold

The three-dimensional structure of methanogen chromosomal protein 1 (MC1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55, has been solved using (1)H NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an alpha helix and five beta strands, arranged as two antiparallel beta sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two beta sheets and the helix side facing the interior of the protein. The MC1 solution structure shows a globular protein with overall dimensions in the range of 34-40 A, which potentially corresponds to a DNA-binding site of 10-12 base pairs. The presumed DNA-binding site is located on the sequence comprising residues K62-P82, which is formed by a part of strands II2 and II3 belonging to the triple-stranded antiparallel beta sheet and a loop flanked by prolines P68 and P76. The tryptophan W74 that is expected to play a key role in the DNA-binding according to photocross-linking experiments was found completely exposed to the solvent, in a good position to interact with DNA. The overall fold of MC1, characterized by its linking beta-beta-alpha-beta-beta-loop-beta, is different from other known DNA-binding proteins. Its structure suggests a different DNA-binding mode than those of the histone-like proteins HU or HMGB. Thus, MC1 may be classified as a member of a new family.     

Clusters in alpha/beta barrel proteins: implications for protein structure, function, and folding: a graph theoretical approach

The alpha/beta barrel fold is adopted by most enzymes performing a variety of catalytic reactions, but with very low sequence similarity. In order to understand the stabilizing interactions important in maintaining the alpha/beta barrel fold, we have identified residue clusters in a dataset of 36 alpha/beta barrel proteins that have less than 10% sequence identity within themselves. A graph theoretical algorithm is used to identify backbone clusters. This approach uses the global information of the nonbonded interaction in the alpha/beta barrel fold for the clustering procedure. The nonbonded interactions are represented mathematically in the form of an adjacency matrix. On diagonalizing the adjacency matrix, clusters and cluster centers are obtained from the highest eigenvalue and its corresponding vector components. Residue clusters are identified in the strand regions forming the beta barrel and are topologically conserved in all 36 proteins studied. The residues forming the cluster in each of the alpha/beta protein are also conserved among the sequences belonging to the same family. The cluster centers are found to occur in the middle of the strands or in the C-terminal of the strands. In most cases, the residues forming the clusters are part of the active site or are located close to the active site. The folding nucleus of the alpha/beta fold is predicted based on hydrophobicity index evaluation of residues and identification of cluster centers. The predicted nucleation sites are found to occur mostly in the middle of the strands. Proteins 2001;43:103-112.     

On the derivation of propensity scales for predicting exposed transmembrane residues of helical membrane proteins

Helical membrane proteins (HMPs) play a crucial role in diverse physiological processes. Given the difficulty in determining their structures by experimental techniques, it is desired to develop computational methods for predicting the burial status of transmembrane residues. Deriving a propensity scale for the 20 amino acids to be exposed to the lipid bilayer from known structures is central to developing such methods. A fundamental problem in this regard is what would be the optimal way of deriving propensity scales. Here, we show that this problem can be reformulated such that an optimal scale is straightforwardly obtained in an analytical fashion. The derived scale favorably compares with others in terms of both algorithmic optimality and practical prediction accuracy. It also allows interesting insights into the structural organization of HMPs. Furthermore, the presented approach can be applied to other bioinformatics problems of HMPs, too. All the data sets and programs used in the study and detailed primary results are available upon request.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.     

Asymmetric amino acid compositions of transmembrane beta-strands

In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane beta-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a beta-strand. This new characteristic of beta-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane beta-barrels.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

The red cell band 3 protein: its role in anion transport

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60% of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.