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The crystal structure of the 32-kDa catalytic domain of the Streptomyces lividans xylanase A was solved by molecular isomorphous replacement methods and subsequently refined at 2.6-A resolution to a conventional crystallographic R factor of 0.21. This is the first successful structure determination of a member of the F family of endo-beta-1,4-D-glycanases. Unlike the recently determined xylanases of the G family (Wakarchuk, W. W., Campbell, R. L., Sung, W. L., Davoodi, J., and Yaguchi, M. (1994) Protein Sci. 3, 467-475), where the catalytic domains have a unique beta-sheet structure, the 32-kDa domain of the S. lividans xylanase A is folded into a complete (alpha/beta)8 barrel, the first such fold observed among beta-1,4-D-glycanases. The active site is located at the carbonyl end of the beta barrel. The crystal structure supports the earlier assignment of Glu-128 and Glu-236 as the catalytic amino acids (Moreau, A., Roberge, M., Manin, C., Shareck, F., Kluepfel, D., and Morosoli, R. (1994) Biochem. J., in press).

To understand the nature of the thyroid hormone binding site, we characterized the binding of 3,3',5-triiodo-L-thyronine (T3) and its analogues to eight naturally occurring mutated human beta 1 thyroid hormone receptors (h-TR beta 1). The mutant receptors were derived from patients with the syndrome of generalized thyroid hormone resistance, and each has a point mutation in the hormone binding domain (KT, R338W; TP, L450H; IR, D322H; NN, G347E; AH, P453H; OK, M442V; RL, F459C; and ED, A317T). Compared to the wild-type h-TR beta 1, binding of T3 was reduced by as much as 97% for the mutants. The order of binding affinity of wild-type h-TR beta 1 to the analogues is T3 > D-T3 > L-thyroxine > 3,5-diiodo-L-thyronine > 3,3',5'-triiodo-L-thyronine. The mutant receptors showed essentially the same order of reduced affinities for the analogues, but the amounts of the reductions varied in each case. These results suggest specific local interactions (interplay) of analogues with the mutated residues in the receptors. On the basis of these data and a putative structure of the hormone binding domains as an eight-stranded alpha/beta barrel, we propose the location of the hormone in the binding site of h-TR beta 1. Ionic bonds anchor the hormone's alanine side chain to loop 4 of the 8-fold alpha/beta barrel. The phenyl ring lies across the amino-terminal face of the domain with the phenoxy ring pointing downward into the barrel interacting with beta-strand 8 on the opposite side.(ABSTRACT TRUNCATED AT 250 WORDS)

Outer membrane proteins are the proteins found in the outer membranes of bacteria, mitochondria and chloroplasts. These proteins adopt a beta barrel structure. There are thousands of outer membrane beta barrels reported in genomic databases with approximately 2-3% of the genes in gram-negative bacteria encoding these proteins. Of the non-redundant outer membrane protein structures in the Protein Data Bank, half have been solved over the past 5 years. This influx of information provides new opportunities for understanding the chemistry of these proteins.Using a structural bioinformatics approach, we have determined a strong asymmetry in the charge distribution of these proteins. For the outward-facing amino acids of the beta barrel within regions of similar amino acid density for both membrane leaflets, the external side of the membrane contains more than three times the number of charged amino acids as the internal side of the membrane. Moreover, the lipid bilayer of the outer membrane is asymmetric, and the overall preference for amino acid types to be in the external leaflet of the membrane correlates roughly with the hydrophobicity of the membrane lipids. This preference is demonstrably related to the difference in lipid composition of the external and internal leaflets of the membrane. The charge asymmetry of proteins in the outer membrane has important implications for how we understand the mechanism of outer membrane protein insertion.

Transmembrane β-barrel (TMB) proteins are a special class of transmembrane proteins which play several key roles in human body and diseases. Due to experimental difficulties, the number of TMB proteins with known structures is very small. Over the years, a number of learning-based methods have been introduced for recognition and structure prediction of TMB proteins. Most of these methods emphasize on homology search rather than any biological or chemical basis. We present a novel graph-theoretic model for classification and structure prediction of TMB proteins. This model folds proteins based on energy minimization rather than a homology search, avoiding any assumption on availability of training dataset. The ab initio model presented in this paper is the first method to allow for permutations in the structure of transmembrane proteins and provides more structural information than any known algorithm. The model is also able to recognize β-barrels by assessing the pseudo free energy. We assess the structure prediction on 42 proteins gathered from existing databases on experimentally validated TMB proteins. We show that our approach is quite accurate with over 90% F-score on strands and over 74% F-score on residues. The results are comparable to other algorithms suggesting that our pseudo-energy model is close to the actual physical model. We test our classification approach and show that it is able to reject β-helical bundles with 100% accuracy and β-barrel lipocalins with 97% accuracy.

β-barrel membrane proteins play an important role in controlling the exchange and transport of ions and organic molecules across bacterial and mitochondrial outer membranes. They are also major regulators of apoptosis and are important determinants of bacterial virulence. In contrast to β-helical membrane proteins, their evolutionary pattern of residue substitutions has not been quantified, and there are no scoring matrices appropriate for their detection through sequence alignment. Using a Bayesian Monte Carlo estimator, we have calculated the instantaneous substitution rates of transmembrane domains of bacterial β-barrel membrane proteins. The scoring matrices constructed from the estimated rates, called bbTM for β-barrel Transmembrane Matrices, improve significantly the sensitivity in detecting homologs of β-barrel membrane proteins, while avoiding erroneous selection of both soluble proteins and other membrane proteins of similar composition. The estimated evolutionary patterns are general and can detect β-barrel membrane proteins very remote from those used for substitution rate estimation. Furthermore, despite the separation of 2-3 billion years since the proto-mitochondrion entered the proto-eukaryotic cell, mitochondria outer membrane proteins in eukaryotes can also be detected accurately using these scoring matrices derived from bacteria. This is consistent with the suggestion that there is no eukaryote-specific signals for translocation. With these matrices, remote homologs of β-barrel membrane proteins with known structures can be reliably detected at genome scale, allowing construction of high quality structural models of their transmembrane domains, at the rate of 131 structures per template protein. The scoring matrices will be useful for identification, classification, and functional inference of membrane proteins from genome and metagenome sequencing projects. The estimated substitution pattern will also help to identify key elements important for the structural and functional integrity of β-barrel membrane proteins, and will aid in the design of mutagenesis studies.

β-Lactoglobulin is a small globular protein found in ruminant milk and many, but not all, other mammals. The cow protein is the major whey protein synthesized as a 180-amino-acid polypeptide from which an 18-residue signal peptide is removed on secretion. Genetic variants involving a few amino acid positions have been reported for many species. The protein is stable between pH 3 and 7 but undergoes conformational change and unfolding as the pH is raised further. The structure is a conical barrel, open at one end, comprising eight antiparallel β-strands with a three-turn α-helix on the outer surface of the barrel. Whereas the ruminant forms of the protein are dimeric, those from other species appear to be monomeric. The protein belongs to a widespread family of small, mostly secretory proteins called the lipocalins, as all members appear to carry small hydrophobic ligands internally. The function of the protein is unknown, although it is a good nutritional source of amino acids, and a transport role of some kind has been suggested. β-Lactoglobulin binds a wide variety of ligands and has been studied intensively not only as a model protein but also because it affects milk stability during commercial processing, its five cysteine residues allowing disulfide interchange to play a key role.

Using site-directed mutagenesis in conjunction with NMR structural data on the adhesion domain of human CD2, we have defined the binding region for CD58. Previous structural studies of rat and human CD2 indicate that this adhesion domain is immunoglobulin-like. Here we report that the CD58 binding site is a well-circumscribed, charged surface area covering approximately 770 A2 on the AGFCC'C" face of the CD2 beta barrel. This site contains beta-strand residues in the carboxyl-terminal half of the F strand (including Lys-82 and Tyr-86), the top of the C strand (Asp-32 and Lys-34), and the C' strand (Gln-46), which are all solvent exposed. In addition, several exposed residues on the FG loop (Gly-90, Lys-91, Asn-92, and Val-93), the CC' loop (Lys-41 and Lys-43), and the C'C" loop (Arg-48 and Lys-51) form this site. In contrast, neither residues on the more peripheral G and C" strands of the same CD2 surface nor residues on B, E, and D strands of the opposite face are involved in CD58 binding. This CD58 binding site is predicted to lie most distal to the T-lymphocyte surface membrane, with ready access to CD58 on the surface of the opposing antigen-presenting cell.

In mammals, the hydroperoxidation of arachidonic acid by lipoxygenases leads to the formation of leukotrienes and lipoxins, compounds that mediate inflammatory responses. Lipoxygenases are dioxygenases that contain a nonheme iron and are present in many animal cells. Soybean lipoxygenase-1 is a single-chain, 839-residue protein closely related to mammalian lipoxygenases. The structure of soybean lipoxygenase-1 solved to 2.6 angstrom resolution shows that the enzyme has two domains: a 146-residue beta barrel and a 693-residue helical bundle. The iron atom is in the center of the larger domain and is coordinated by three histidines and the COO- of the carboxyl terminus. The coordination geometry is nonregular and appears to be a distorted octahedron in which two adjacent positions are not occupied by ligands. Two cavities, in the shapes of a bent cylinder and a frustum, connect the unoccupied positions to the surface of the enzyme. The iron, with two adjacent and unoccupied positions, is poised to interact with the 1,4-diene system of the substrate and with molecular oxygen during catalysis.

A series of single and double amino acid replacements in four beta strands of the alpha subunit of tryptophan synthase from Salmonella typhimurium, and alpha/beta barrel protein, was made to study the interior packing of the barrel and to clarify its folding mechanism. The urea-induced unfolding of the alpha subunit is thought to involve a stable intermediate in which the amino folding unit (residues 1-188; helices 0-5, strands 1-6) remains folded while the carboxy folding unit (residues 189-268; helices 6-8, strands 7-8) becomes disordered [Beasty, A. M., & Matthews, C. R. (1985) Biochemistry 24, 3547; Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586]. Mutations in strands 1 (A18G and A18V), 6 (Y175Q), 7 (L209V), and 8 (G230A, G230V, and I232V) at the interface between these two folding units show that the effects on the stabilities of the native and intermediate conformations critically depend on the site of the replacement. Although all of these mutations decrease the stability of the native conformation, only the replacements in strand 6, Y175Q, and possibly strand 8, I232V, also perturb the intermediate. Comparisons of the effects of three pairs of double mutants with the effects of the constituent single mutants on stability show that strands 6 and 7 interact in both the intermediate and native conformations, while strands 1 and 8 interact only in the native conformation. Kinetic studies of unfolding indicate that the interactions which occur in the native conformation arise in the preceding transition state. These results demonstrate that the carboxy folding unit adopts an organized structure in the intermediate, contrary to our previous interpretation. The general implication is that the state of folding of one segment of a protein can depend on the presence of another, more stable element of structure.

SH3 domains are found in proteins associated with receptor tyrosine kinase signal transduction complexes. The solution structure of the SH3 domain of the 85 kd regulatory subunit of phosphatidylinositol 3-kinase is shown to be a compact beta barrel consisting of five beta strands arranged in two beta sheets of three and two strands. The structure is similar to that of chicken brain alpha spectrin but represents a distinct class of SH3 domain, with an insertion between the second and third beta strands that may influence binding specificity. 1H chemical shift changes induced by complex formation with a synthetic peptide derived from the SH3-binding protein dynamin, together with amino acid sequence comparisons, suggest that the ligand-binding site consists of a hydrophobic surface flanked by two charged loops.

Several computational methods exist for the identification of transmembrane beta barrel proteins (TMBs) from sequence. Some of these methods also provide the transmembrane (TM) boundaries of the putative TMBs. The aim of this study is to (1) derive the propensities of the TM residues to be exposed to the lipid bilayer and (2) to predict the exposure status (i.e. exposed to the bilayer or hidden in protein structure) of TMB residues. Three novel propensity scales namely, BTMC, BTMI and HTMI were derived for the TMB residues at the hydrophobic core region of the outer membrane (OM), the lipid-water interface regions of the OM, and for the helical membrane proteins (HMPs) residues at the lipid-water interface regions of the inner membrane (IM), respectively. Separate propensity scales were derived for monomeric and functionally oligomeric TMBs. The derived propensities reflect differing physico-chemical properties of the respective membrane bilayer regions and were employed in a computational method for the prediction of the exposure status of TMB residues. Based on the these propensities, the conservation indices and the frequency profile of the residues, the transmembrane residues were classified into buried/exposed with an accuracy of 77.91% and 80.42% for the residues at the membrane core and the interface regions, respectively. The correlation of the derived scales with different physico-chemical properties obtained from the AAIndex database are also discussed. Knowledge about the residue propensities and burial status will be useful in annotating putative TMBs with unknown structure.

Among all beta-barrel pores, staphylococcal alpha-hemolysin (alphaHL), a heptameric transmembrane protein of known high-resolution crystal structure, features a high stability in planar lipid bilayers under a wide range of harsh experimental conditions. Here, we employed single-channel electrical recordings and standard protein engineering to explore the impact of two distant charge reversals within the interior of the beta-barrel part of the pore. The charge reversals were replacements of lysines with aspartic acids. A charge reversal within the structurally stiff region of the beta barrel near the pore constriction reduced the open-state current of the pore, but produced a quiet pore, showing current fluctuation-free channel behavior. In contrast, a charge reversal on the trans entrance, within the structurally flexible glycine-rich turn of the beta barrel, increased the open-state current and produced gating activity of the pore in the form of large-amplitude and frequent current fluctuations. Remarkably, cumulative insertion of the two distant charge reversals resulted in a large-amplitude permanent blockade of the beta barrel, as judged by both single-channel and macroscopic current measurements. The results from this work suggest that these distant charge reversals are energetically coupled, producing different impacts on the ionic transport, the unitary conductance and the open-state probability of the pore.

We present BTMX (Beta barrel TransMembrane eXposure), a computational method to predict the exposure status (i.e. exposed to the bilayer or hidden in the protein structure) of transmembrane residues in transmembrane beta barrel proteins (TMBs). BTMX predicts the exposure status of known TM residues with an accuracy of 84.2% over 2,225 residues and provides a confidence score for all predictions. Predictions made are in concert with the fact that hydrophobic residues tend to be more exposed to the bilayer. The biological relevance of the input parameters is also discussed. The highest prediction accuracy is obtained when a sliding window comprising three residues with similar Cα- Cβvector orientations is employed. The prediction accuracy of the BTMX method on a separate unseen non-redundant test dataset is 78.1%. By employing out-pointing residues that are exposed to the bilayer, we have identified various physico-chemical properties that show statistically significant differences between the beta strands located at the oligomeric interfaces compared to the non-oligomeric strands. The BTMX web server generates colored, annotated snake-plots as part of the prediction results and is available under the BTMX tab at . Exposure status prediction of TMB residues may be useful in 3D structure prediction of TMBs.

We have developed an effective pathway for the prediction and characterization of novel transmembrane β-barrel proteins. The Freeman–Wimley algorithm, which is a highly accurate prediction method based on the physicochemical properties of experimentally characterized transmembrane β barrel (TMBB) structures, was used to predict TMBBs in the genome of Salmonella typhimurium LT2. The previously uncharacterized product of gene yshA was tested as a model for validating the algorithm. YshA is a highly conserved 230-residue protein that is predicted to have 10 transmembrane β-strands and an N-terminal signal sequence. All of the physicochemical and spectroscopic properties exhibited by YshA are consistent with the prediction that it is a TMBB. Specifically, recombinant YshA localizes to the outer membrane when expressed in Escherichia coli ; YshA has a β-sheet-rich secondary structure with stable tertiary contacts in the presence of detergent micelles or when reconstituted into a lipid bilayer. When in a lipid bilayer, YshA forms a membrane-spanning pore with an effective radius of ~ 0.7 nm. Taken together, these data substantiate the predictions made by the Freeman–Wimley algorithm by showing that YshA is a TMBB protein.

The interaction of ferriheme proteins with nitrite has recently attracted interest as a source for NO or other nitrogen oxides in mammalian physiology. However, met-hemoglobin (metHb), which was suggested as a key player in this process, does not convert nitrite unless small amounts of NO are added in parallel. We have recently reported that, in contrast, nitrophorins (NPs) convert nitrite as the sole substrate to form NO even at pH 7.5, which is an unprecedented case among ferrihemes [He, C., and Knipp, M. (2009) J. Am. Chem. Soc. 131, 12042-12043]. NPs, which comprise a class of unique heme b proteins from the saliva of the blood-sucking insect Rhodnius prolixus, appear in a number of concomitant isoproteins. Herein, the first spectroscopic characterization of the initial complexes of the two isoproteins NP4 and NP7 with nitrite is presented and compared to the data reported for metHb and met-myoglobin (metMb). Because upon nitrite binding, NPs, in contrast to metHb and metMb, continue to react with nitrite, resonance Raman spectroscopy and continuous wave electron paramagnetic resonance spectroscopy were applied to frozen samples. As a result, the existence of two six-coordinate ferriheme low-spin complexes was established. Furthermore, X-ray crystallography of NP4 crystals soaked with nitrite revealed the formation of an eta(1)-N nitro complex, which is in contrast to the eta(1)-O-bound nitrite in metMb and metHb. Stopped-flow kinetic experiments show that although the ligand dissociation constants of NP4 and NP7 (15-190 M(-1)) are comparable to those of metHb and metMb, the rates of ligand binding and release are significantly slower. Moreover, not only the reaction kinetics but also electron paramagnetic resonance spectroscopy reveals notable differences between the two isoproteins.

Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence., OMP85 is an evolutionarily conserved protein necessary for the assembly of integral beta-barrel proteins in bacteria. The authors show that OMP85 forms a channel and recognizes its unfolded subtrates through a C-terminal signature sequence.

Among the two classes of membrane proteins, beta-barrel membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and are often responsible for bacterial virulence. Although membrane proteins comprise approximately one third of all proteins encoded in a genome, they are sparsely represented in the protein structure databank, due to difficulties in experimental structural determination. We have developed a computational method to predict three dimensional structures of beta-barrel membrane proteins from transmembrane sequence segments. For this, we have derived an asymmetric potential function based on detailed combinatorial analysis. In addition, we have developed a model to account for interstrand loop entropy. In a set of 25 non-homologous proteins with known structures, we can successfully predict strand register at 76% accuracy. This is a significant improvement from previous results (44%) and from random chance (7%) [1]. Based on predicted strand registrations, we are now able to predict the three dimensional structure of the transmembrane region of beta-barrel membrane proteins. The average RMSD between predicted and native beta-barrel membrane protein structure is less than 3A°. Our method is general and can be applied to genome-wide structural prediction once sequences of beta-barrel membrane proteins have been identified.[1] Ronald Jackups, Jr. and Jie Liang. Interstrand pairing patterns in beta-barrel membrane proteins: the positive-outside rule, aromatic rescue, and strand registration prediction J Mol Biol. 2005, 354:979-993.

Transmembrane β-barrel (TMB) proteins are embedded in the outer membranes of mitochondria, Gram-negative bacteria, and chloroplasts. These proteins perform critical functions, including active ion-transport and passive nutrient intake. Therefore, there is a need for accurate prediction of secondary and tertiary structures of TMB proteins. A variety of methods have been developed for predicting the secondary structure and these predictions are very useful for constructing a coarse topology of TMB structure; however, they do not provide enough information to construct a low-resolution tertiary structure for a TMB protein. In addition, while the overall structural architecture is well conserved among TMB proteins, the amino acid sequences are highly divergent. Thus, traditional homology modeling methods cannot be applied to many putative TMB proteins. Here, we describe the TMBpro: a pipeline of methods for predicting TMB secondary structure, β-residue contacts, and finally tertiary structure. The tertiary prediction method relies on the specific construction rules that TMB proteins adhere to and on the predicted β-residue contacts to dramatically reduce the search space for the model building procedure.

The structure of the unligated recombinant human cyclophilin A (CyP A) has been refined to an R-factor of 0.18 at 1.63 A resolution. The root-mean-squared deviations of the refined structure are 0.013 A and 2.50 ° from ideal geometries of bond length and bond angle, respectively. Eight antiparallel β-strands of CyP A form a right-handed β-barrel. The structure of CyP A is compared with other members in the antiparallel eight-stranded β-barrel family and with the parallel eight-stranded αβ barrels. Although all known eight-stranded barrels are right-handed, the tilted angle of the strands against the barrel axis varies from 45 ° for retinol binding protein and 49 ° for CyP A to 70 ° for Superoxide dismutase. As a result, the β-barrel of CyP A is not completely superimposable with other members of β-barrels. The structure of CyP A has a unique topology, distinct from other members in the β-barrel family. In addition, CyP A is a closed β-barrel so that neither the immunosuppressive drug cyclosporin A (CsA) nor the proline-containing substrate can bind to the hydrophobic core of the CyP A barrel, while the hydrophobic core of most other barrels is open for ligation. These observations probably indicate that CyP A is neither functionally nor evolutionally related to other β-barrel structures. Details of interactions between solvent molecules and the active site residues of CyP A are illustrated. A water-co-operated mechanism, where the cis ⇌ trans isomerization might possibly consist of (1) transition of the prolyl bond and (2) release of N or C-terminal residues of substrate from CyP, is addressed. The refined structure reveals no disulfide bridges in CyP A. Cys115 is near the CsA site, but unlikely to be directly involved in CsA binding because of steric hindrance from Thr119 and Leu122. This geometry probably rules out any mechanisms involving a tetrahedral intermediate formed between cysteine and substrate during cis ⇌ trans isomerization.

Aldose reductase, which catalyzes the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of a wide variety of aromatic and aliphatic carbonyl compounds, is implicated in the development of diabetic and galactosemic complications involving the lens, retina, nerves, and kidney. A 1.65 angstrom refined structure of a recombinant human placenta aldose reductase reveals that the enzyme contains a parallel beta 8/alpha 8-barrel motif and establishes a new motif for NADP-binding oxidoreductases. The substrate-binding site is located in a large, deep elliptical pocket at the COOH-terminal end of the beta barrel with a bound NADPH in an extended conformation. The highly hydrophobic nature of the active site pocket greatly favors aromatic and apolar substrates over highly polar monosaccharides. The structure should allow for the rational design of specific inhibitors that might provide molecular understanding of the catalytic mechanism, as well as possible therapeutic agents.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.

Simulations of the folding pathways of two large alpha/beta proteins, the alpha subunit of tryptophan synthase and triose phosphate isomerase, are reported using the knight's walk lattice model of globular proteins and Monte Carlo dynamics. Starting from randomly generated unfolded states and with no assumptions regarding the nature of the folding intermediates, for the tryptophan synthase subunit these simulations predict, in agreement with experiment, the existence and location of a stable equilibrium intermediate comprised of six beta strands on the amino terminus of the molecule. For the case of triose phosphate isomerase, the simulations predict that both amino- and carboxyl-terminal intermediates should be observed. In a significant modification of previous lattice models, this model includes a full heavy atom side chain description and is capable of representing native conformations at the level of 2.5- to 3-A rms deviation for the C alpha positions, as compared to the crystal structure. With a well-balanced compromise between accuracy of the protein description and the computer requirements necessary to perform simulations spanning biologically significant amounts of time, the lattice model described here brings the possibility of studying important biological processes to present-day computers.

This study investigates the catalytic and allosteric roles of a flexible loop in the tryptophan synthase alpha 2 beta 2 complex. This loop connects helix 6 and strand 6 in the alpha subunit, an 8-fold alpha/beta barrel polypeptide. We have engineered three mutations in this disordered loop: a deletion of residues 185-187 and the replacement of threonine 183 by serine (T183S) or by alanine (T183A). Position 183 is a site of an inactivating mutation identified by Yanofsky's group (Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton, B. C. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 296-298). The three engineered alpha subunits form stable, stoichiometric alpha 2 beta 2 complexes with the beta subunit which bind alpha and beta subunit ligands. Although changing threonine 183 to serine has little effect on the enzymatic properties, changing threonine 183 to alanine or deleting residues 185-187 results in a 50-fold reduction in the intrinsic activity of the alpha subunit alone and in the alpha site activity of the alpha 2 beta 2 complex. The latter two mutations profoundly alter the way in which the alpha subunit modulates the spectral properties and the activities of the wild-type beta subunit. These mutations also eliminate the effects of alpha subunit ligands on the beta subunit. Although the beta subunit ligand, L-serine, greatly stabilizes the wild-type alpha 2 beta 2 complex to dissociation and to proteolysis, L-serine stabilizes the T183A alpha 2 beta 2 complex weakly or not at all. Our findings suggest that the hydroxyl residue at position 183 and the adjacent residues in the alpha subunit loop play critical roles in the reciprocal communication between the alpha and beta subunits in the alpha 2 beta 2 complex. The results also help to explain how the wild-type alpha subunit or ammonium ion modulates the activities of the beta subunit.

Previous crystallographic analyses of the Kunitz inhibitors from soybean, Erythrina caffra and wheat, the interleukins-1β and 1α and the acidic and basic fibroblast growth factors have shown that they contain a most unusual fold. It is formed by six two-stranded hairpins. Three of these form a barrel structure and the other three are in a triangular array that caps the barrel. The arrangement of the secondary structures gives the molecules a pseudo 3-fold axis. Although the different proteins have very similar structures, many of their sequences have no significant similarities overall. The structural determinants of this fold are described and discussed in this paper. The barrels in the different proteins have the same geometrical features: six strands tilted at 56 ° to the barrel axis; a barrel diameter of 16 A, and the β-sheet hydrogen bonded so that it is staggered with a shear number of 12. These features fit McLachlan's equations for ideal barrels formed by β-sheets. The wide diameter of the barrels is filled by layers of residues that, while not identical in the different proteins, are, in almost all cases, large. The structure of the triangular array of hairpins is determined by the coiling of the strands and the packing of hairpin residues against each other and against residues from the interior of the barrel. The major sequence requirements of this fold are large or medium hydrophobic resiudes at 18 buried sites. In the different structures the total volume of these residues is 3000(±120) A 3 . The polyhedron model of protein architecture is used to demonstrate that the main, and in particular the symmetrical, features of this fold arise from the ideal and equal packing of six hairpins, modified only slightly to form hydrogen bonds between the hairpins.

A lingering issue in the area of protein engineering is the optimal design of beta motifs. In this regard, the framework provided by intestinal fatty acid binding protein (IFABP) was successfully chosen to explore the consequences on structure and function of the redesign of natural motifs. A truncated form of IFABP (Delta 98 Delta) served to illustrate the nonintuitive notion that the integrity of the beta-barrel can indeed be compromised with no effect on the ability to attain a native-like fold. This is most likely the outcome of the key role played by the preservation of essential core residues. In the search for the minimal structural determinants of this fold, Delta 98 Delta offered room for further intervention. A dissection of this protein leads to a new abridged variant, Delta 78 Delta, containing 60% of the amino acids of IFABP. Spectroscopic analyses indicate that Delta 78 Delta retains substantial beta-sheet content and preserves tertiary interactions, displaying cooperative unfolding and binding activity. Most strikingly, this construct adopts a remarkably stable dimeric structure in solution. This phenomenon takes advantage of the inherent structural plasticity of this motif, likely profitting from edge-to-edge interactions between beta-sheets, whereas avoiding the most commonly occurring outcome represented by aggregation. Fil: Franchini, Gisela Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Curto, Lucrecia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Delfino, Jose Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Intrinsically fluorescent proteins (FPs) exhibit broad variations of absorption and emission colors and are available for different imaging applications. The physical cause of the absorption wavelength change from 540 nm to 590 nm in the Fruits series of red FPs has been puzzling because the mutations that cause the shifts do not disturb the π-conjugation pathway of the chromophore. Here we use two-photon absorption measurements to show that the different colors can be explained by quadratic Stark effect due to variations of the strong electric field within the beta barrel. This model brings simplicity to a bewildering diversity of fluorescent protein properties, and it suggests a new way to sense electrical fields in biological systems.

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel beta-strands forming a beta-barrel with an alpha-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP.biliverdin IX(gamma) and FluA.fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the beta-barrel as well as adjoining beta-strand segments. An "induced fit" became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse (15)N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA.fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called "anticalins".

Proteins of the TpsB/Omp85 superfamily are involved in protein transport across, or assembly into, the outer membrane of Gram-negative bacteria, and their distant eukaryotic relatives exert similar functions in chloroplasts and mitochondria. The X-ray structure of one TpsB transporter, FhaC, provides the bases to decipher the mechanisms of action of these proteins. With two POTRA domains in the periplasm, a transmembrane β barrel and a large loop harboring a functionally important motif, FhaC epitomizes the conserved features of the super-family.

Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P2(1) with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 A resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P2(1) crystal form uncovered major conformational changes (i) in beta-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the beta-barrel and (iii) in the extended C-terminal segment, which is attached to the beta-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family.

Autotransporters are outer membrane proteins produced by Gram-negative bacteria that are involved in virulence and consist of two domains, an N-terminal “passenger domain” and a C-terminal “β-domain”. Passenger domains are secreted to the cell surface while β-domains are predicted to form β-barrel structures in the outer membrane. In some autotransporters, the secreted passenger domain is released from the β-domain by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7 A structure of the post-cleavage state of the β-domain of EspP, an autotransporter produced by E. coli O157:H7. The structure consists of a 12-stranded β-barrel with the passenger / β-domain cleavage junction located inside the barrel pore, approximately mid-way between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the β-domain after cleavage, one conferring increased stability on the β-domain and another restricting access to the barrel pore. The location of the cleavage site within the beta barrel has implications for secretion of the passenger domain across the outer membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Computational and statistical analysis has formed a large component of the biophysical efforts put forth to understand protein structure and function, due to the diversity and complexity of their structure. Outer membrane proteins form a diverse and complex set of proteins. Of these, porins that allow passage of molecules across the membrane interface have been analyzed here from a biophysical and structural perspective. The objective of this study is to analyze the structural organization of porins using atomic temperature factor as a parameter. Generally atomic temperature factors of molecules from crystal structures indicate the degree of mobility or disorder seen in the crystal structure. As good crystal structures have lesser possibilities of errors so there is lesser chance that errors are playing roles in temperature factors. Structures of six porins (four 16-stranded beta barrel porins and two 8-stranded beta barrel porins) were taken from the PDB for the analysis based on resolution and R-factor. Programs and scripts were written for extracting the temperature factors for the beta strands, loops and turns so that the analysis could be done for different atom-types and residue-types. The residue distribution and mobility distribution was found to be characteristic of each of the porins. The mobility and residue distribution amongst the secondary structural elements were found to follow the level of homology at the sequence and structural level. The loops that had defined functional roles in structural terms were found to have lower temperature factors than the other loops. The turn regions that are thought to face the periplasmic region in the cell, showed higher temperature factors. For both the 16 stranded and the 8-stranded barrels it was found one part of the barrel (the lower wall or 'inner' wall comprising the trimer interface in the case of the 16-stranded barrels) was more rigid and the other half of the barrel (the higher or 'outer' wall) showed more mobility as seen from the temperature factors. This seems to be an intrinsic structural component of the beta barrels.

Amyloidβ(Aβ) annular protofibrils (APFs) have been described where the structure is related to that ofβbarrel pore-forming bacterial toxins and exhibits cellular toxicity. To investigate the relationship of AβAPFs to disease and their ultrastructural localization in brain tissue, we conducted a pre-embedding immunoelectron microscopic study using anti-annular protofibril antiserum. We examined brain tissues of young- and old-aged amyloid precursor protein transgenic mice (APP23), neprilysin knockout APP23 mice, and nontransgenic littermates.αAPF-immunoreactions tended to be found (1) on plasma membranes and vesicles inside of cell processes, but not on amyloid fibrils, (2) with higher density due to aging, APP transgene, and neprilysin deficiency, and (3) with higher positive rate at synaptic compartments in aged APP23, especially in neprilysin knockout APP23 mice. These findings imply that APFs are distinct from amyloid fibrils, interact with biological membranes, and might be related to synaptic dysfunction in Alzheimer model mouse brains.

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.

During the past few years one of the most astonishing findings in the field of virology has been the realization that viruses that infect hosts from all three domains of life are often structurally similar. The recent burst of structural information points to a need to create a new way to organize the virosphere that, in addition to the current classification, would reflect relationships between virus families. Using the vertical beta-barrel major capsid proteins and ATPases related to known viral genome-packaging ATPases as examples, we can now re-evaluate the classification of viruses and virus-like genetic elements from a structural standpoint.

Outer membrane beta-stranded porins form a diverse and complex set of proteins which allow passage of molecules across the membrane interface have been analyzed here from a biophysical and structural perspective using atomic temperature factors or B-factors. Generally atomic temperature factors of molecules from crystal structures indicate the degree of mobility or disorder seen in the crystal structure. Structures of six porins (four 16 stranded beta barrel porins and two 8 stranded beta barrel porins) were taken from the PDB for the analysis based on resolution (better than 3.0 Å) and R-factor (< 0.23). The residue distribution and mobility distribution was found to be characteristic of each of the porins. The mobility and residue distribution amongst the secondary structural elements were found to follow the level of homology at the sequence and structural level. The loops (L2 and L3) that had defined functional roles in structural terms were found to have lower temperature factors than the other loops. The turn regions that are thought to face the periplasmic region in the cell, showed higher temperature factors. For both the 16 stranded and the 8 stranded barrels it was found one part of the barrel (the lower wall or 'inner' wall comprising the trimer interface in the case of the 16 stranded barrels) was more rigid and the other half of the barrel (the higher or 'outer' wall) showed more mobility as seen from the temperature factors. This seems to be an intrinsic structural component of the beta barrels.

Over the past decade, understanding of the structure and function of membrane proteins has advanced significantly as well as how their detailed characterization can be approached experimentally. Detergents have played significant roles in this effort. They serve as tools to isolate, solubilize, and manipulate membrane proteins for subsequent biochemical and physical characterization. Combination of detergents and various separation methods coupled with mass spectrometry technology e.g. MALDI-TOF/TOF and nano-HPLC-ESI-Q-TOF/MS/MS is now possible to examine the expression of membrane proteins. This study for establishing separation methods of membrane proteins on two modified gel-electrophoresis (16-BAC and BN-PAGE subsequently with SDS-PAGE) could make it likely that the components of membranes become increasingly amenable to identification and characterization. To study the structure (complexes) and function of membrane proteins, we must first pre-fractionate enriched membrane proteins, or isolate and purify membrane complexes. Such proteins can be solubilized by high-salt solutions or detergents, which have affinity both for hydrophobic groups and for water. Due to a preponderance of binding detergents over hydrophobic regions, when integral proteins are exposed on aqueous solution, these protein molecules are prevented from aggregation and maintained their native conformation. Subsequently, diverse kinds of eletrophoretic analysis combined with mass spectrometry have been applied with site specific (tripsin, chymotrypsin, CNBr and Asp-N) enzymes. The final goal is to enable high-throughput analysis of ion-channel proteins and major neurotransmitter receptor complexes within central nervous system by an electrophoretic method allowing quantification with subsequent unambiguous protein identification.

In eukaryotes, a voltage-dependent anion channel (VDAC) facilitates diffusion of metabolites across the mitochondrial outer membrane. Closure of the human isoform, VDAC-1, leads to mitochondrial apoptosis. Hiller et al . have determined the nuclear magnetic resonance solution structure of VDAC-1 in detergent micelles. Whereas known beta barrel structures have an even number of strands, VDAC-1 has a 19-stranded barrel with parallel pairing of the first and last strands. Chemical shift data were used to map interaction sites for the substrates NADH and cholesterol and for the antiapoptotic protein Bcl-x L that opens the channel and thus inhibits apoptosis. The structure provides a framework for reanalysis of existing biochemical and biophysical data. S. Hiller, R. G. Garces, T. J. Malia, V. Y. Orekhov, M. Colombini, G. Wagner, Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321 , 1206-1210 (2008). [Abstract][Full Text]

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.

The voltage-dependent anion channel (VDAC) mediates trafficking of small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC also interacts with antiapoptotic proteins from the Bcl-2 family, and this interaction inhibits release of apoptogenic proteins from the mitochondrion. We present the nuclear magnetic resonance (NMR) solution structure of recombinant human VDAC-1 reconstituted in detergent micelles. It forms a 19-stranded β barrel with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a beltlike fashion. In the presence of cholesterol, recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to those of the native protein. NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-x L , for reduced β–nicotinamide adenine dinucleotide, and for cholesterol. Bcl-x L interacts with the VDAC barrel laterally at strands 17 and 18.

Beta-barrel membrane proteins (MP) are found in Gram-negative bacteria, mitochondria and chloroplasts. They play important roles in metabolism of bacteria, where they are involved in transport of solutes in and out of the cell. Beta-barrel proteins may also act as proteases, lipases and may be important for cell-cell adhesion. Currently, there are about 30 non-redundant solved structures of beta-barrels. Although the number of b-barrel folds is fairly small, it is possible to expand the amount of available structural information by homology modeling using existing structures as templates. The scope of structure prediction may be widened by finding remote homologues of the existing structures. To improve the sensitivity of the database searches and the quality of sequence alignments, we first study evolutionary history of transmembrane segments of 7 beta-barrel membrane proteins by estimating substitution rates with a Bayesian Monte Carlo approach. Next, we calculate amino acid substitution matrices, beta-barrel Transmembrane scoring Matrices (bbTM), specifically tuned for TM regions, which can be used to detect remote homologues. We then test bbTM matrices by comparing their performance with membrane-protein derived scoring matrices PHAT and SLIM. Our results demonstrate that bbTM matrices have higher selectivity towards transmembrane beta-barrel proteins and may be used with higher confidence in database searches for remote homologues of this class of proteins.

Methyl parathion hydrolase mph gene was cloned from a novel bacterium Pseudomonas stutzeri HS-D36. The phylogenetic tree was constructed and the structure was predicted by bioinformatics. Results showed MPH protein was 99.0% similar to MPD protein (from pseudomonas sp. WBC-3) and it belonged to metallo-beta-lactamases family. There is an alpha/beta barrel structure in MPH structure. Two independent subunits comprise a homodimer, each subunit is composed of an active metal center (Zn2+ and Cd2+). Homology modeling of MPH was constructed based on the crystal structure of MPD (PDBID.1P9E). Results showed that three mutation amino acid residues in MPH protein were different from MPD, but the function of MPH protein did not change compared to MPD, this indicated the three residues was not important to the activity of MPH enzyme.

Staphylococcal leucotoxins, leucocidins, and gamma-hemolysins are bicomponent beta-barrel pore-forming toxins (beta-PFTs). Their production is associated with several clinical diseases. They have cytotoxic activity due to the synergistic action of a class S component and a class F component, which are secreted as water-soluble monomers and form hetero-oligomeric transmembrane pores, causing the lysis of susceptible cells. Structural information is currently available for the monomeric S and F proteins and the homoheptamer formed by the related alpha-hemolysin. These structures illustrate the start and end points in the mechanistic framework of beta-PFT assembly. Only limited structural data exist for the intermediate stages, including hetero-oligomeric complexes of leucotoxins. We investigated the protein-protein interactions responsible for maintaining the final bipartite molecular architecture and describe here the high-resolution crystal structure and low-resolution solution structure of a site-specific cross-linked heterodimer of gamma-hemolysin (HlgA T28C-HlgB N156C), which were solved by X-ray crystallography and small angle X-ray scattering, respectively. These structures reveal a molecular plasticity of beta-PFTs, which may facilitate the transition from membrane-bound monomers to heterodimers.

Originally published in: Protein Folding Handbook. Part I. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30784-2 The sections in this article are Introduction Thermodyamics of Residue Partitioning into Lipid Bilayers Stability of β-Barrel Proteins Stability of Helical Membrane Proteins Helix and Other Lateral Interactions in Membrane Proteins The Membrane Interface as an Important Contributor to Membrane Protein Folding Membrane Toxins as Models for Helical Membrane Protein Insertion Mechanisms of β-Barrel Membrane Protein Folding Experimental Protocols SDS Gel Shift Assay for Heat-modifiable Membrane Proteins Reversible Folding and Unfolding Protocol Using OmpA as an Example Tryptophan Fluorescence and Time-resolved Distance Determination by Tryptophan Fluorescence Quenching TDFQ Protocol for Monitoring the Translocation of Tryptophans across Membranes Circular Dichroism Spectroscopy Fourier Transform Infrared Spectroscopy Protocol for Obtaining Conformation and Orientation of Membrane Proteins and Peptides by Polarized ATR-FTIR Spectroscopy Acknowledgments Keywords: protein folding; membrane proteins; beta barrel proteins; circular dichroism; protein stability

Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and bacterial virulence. In addition, beta-barrel membrane proteins increasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank and computational methods can help to understand their biophysical principles. We have developed a novel computational method to predict the 3D structure of beta-barrel membrane proteins using evolutionary coupling (EC) constraints and a reduced state space. Combined with an empirical potential function, we can successfully predict strand register at >80% accuracy for a set of 49 non-homologous proteins with known structures. This is a significant improvement from previous results using EC alone (44%) [1] and using empirical potential function alone (73%) [2]. Our method is general and can be applied to genome-wide structural prediction.[1] Sikander Hayat, Chris Sander, Debora S. Marks, and Arne Elofsson. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences. PNAS 2015 112 (17) 5413-5418.[2] Hammad Naveed, Yun Xu, Ronald Jackups, Jr., and Jie Liang. Predicting Three-Dimensional Structures of Transmembrane Domains of β-Barrel Membrane Proteins. Journal of the American Chemical Society 2012 134 (3), 1775-1781.

The protein-folding problem is one of the contemporary challenges of biophysics. Knowing how the sequence encodes a native structure will provide insight into how genetic mutations cause disease, how protein sequences evolve and represents a tool to design proteins with novel structures and functions. While the folding of soluble proteins has been studied for decades, studies on membrane proteins are not as common. In this talk, I will present our recent developments in membrane protein stability measurements in vitro as well as their implications in living cells.

We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures. Our approach consists of repeating eight times a unit peptide designed to adopt a 'beta-strand/alpha-helix' pattern. A first 'test' sequence for this structural unit was deduced from a series of parameters defined after an analysis of three natural alpha/beta-barrel proteins and including principally the lengths of the secondary structure elements, the alpha/beta packing and the fitting on average Garnier profiles. The gene encoding this structural unit was synthesized, cloned and expressed in Escherichia coli either as a monomer or as direct repeats of 2-12 units. Preliminary structural characterization of the 7-, 8- and 9-fold unit polypeptides by circular dichroism measurements indicates the presence of the predicted amount of alpha-helix in the three proteins. Further analysis by urea-gradient gel electrophoresis demonstrates that, in the conditions tested, only the 8-fold unit polypeptide forms a compact structure through a cooperative and rapid two-state folding transition involving long-range molecular interactions.

Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.

International audience; The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. Proteins 2007. (c) 2007 Wiley-Liss, Inc.