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An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.

Chitin is second only to cellulose in biomass and it is an important component of many cell wall structures. Several families of enzymes, of distinctly different structure, have evolved to hydrolyze this important polysaccaride. Glycohydrolase family 18 enzymes, chitinases, are characterized by an eight-fold alpha/beta barrel structure; it has representatives among bacteria, fungi, and higher plants. In general these chitinases act through a retaining mechanism in which beta linked polymer is cleaved to release a beta anomer product. Family 19 chitinases are found primarily in plants but some are found in bacteria. Members of this family are related to one another by amino acid sequence, but are unrelated to family 18 proteins. They have a bilobal structure with a high alpha-helical content. Despite any significant sequence homology with lysozymes, structural analysis reveals that family 19 chitinases, together with family 46 chitosanases, are similar to several lysozymes including those from T4-phage and from goose. The structures reveal that the different enzyme groups arose from a common ancestor glycohydrolase antecedent to the procaryotic/eucaryotic divergence. In general, the family 19 enzymes operate through an inverting mechanism.

International audience; FhuA protein facilitates ligand-gated transport of ferrichrome-bound iron across Escherichia coli outer membranes. X-ray analysis at 2.7 A resolution reveals two distinct conformations in the presence and absence of ferrichrome. The monomeric protein consists of a hollow, 22-stranded, antiparallel beta barrel (residues 160-714), which is obstructed by a plug (residues 19-159). The binding site of ferrichrome, an aromatic pocket near the cell surface, undergoes minor changes upon association with the ligand. These are propagated and amplified across the plug, eventually resulting in substantially different protein conformations at the periplasmic face. Our findings reveal the mechanism of signal transmission and suggest how the energy-transducing TonB complex senses ligand binding.

Hydrolysis of the major structural polysaccharides of plant cell walls by the aerobic soil bacterium Pseudomonas fluorescens subsp. cellulosa is attributable to the production of multiple extracellular cellulase and hemicellulase enzymes, which are the products of distinct genes belonging to multigene families. Cloning and sequencing of individual genes, coupled with gene sectioning and functional analysis of the encoded proteins have provided a detailed picture of structure/function relationships and have established the cellulase-hemicellulase system of P. fluorescens subsp. cellulosa as a model for the plant cell wall degrading enzyme systems of aerobic cellulolytic bacteria. Cellulose- and xylan-degrading enzymes produced by the pseudomonad are typically modular in structure and contain catalytic and noncatalytic domains joined together by serine-rich linker sequences. The cellulases include a cellodextrinase; a beta-glucan glucohydrolase and multiple endoglucanases, containing catalytic domains belonging to glycosyl hydrolase families 5, 9, and 45; and cellulose-binding domains of families II and X, both of which are present in each enzyme. Endo-acting xylanases, with catalytic domains belonging to families 10 and 11, and accessory xylan-degrading enzymes produced by P. fluorescens subsp. cellulosa contain cellulose-binding domains of families II, X, and XI, which act by promoting close contact between the catalytic domain of the enzyme and its target substrate. A domain homologous with NodB from rhizobia, present in one xylanase, functions as a deacetylase. Mananase, arabinanase, and galactanase produced by the pseudomonad are single domain enzymes. Crystallographic studies, coupled with detailed kinetic analysis of mutant forms of the enzyme in which key residues have been altered by site-directed mutagenesis, have shown that xylanase A (family 10) has 8-fold alpha/beta barrel architecture, an extended substrate-binding cleft containing at least six xylose-binding pockets and a calcium-binding site that protects the enzyme from thermal inactivation, thermal unfolding, and attack by proteinases. Kinetic studies of mutant and wild-type forms of a mannanase and a galactanase from P. fluorescens subsp. cellulosa have enabled the catalytic mechanisms and key catalytic residues of these enzymes to be identified.

Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.

The structure and conformational behaviour of a vector peptide, designed by association of a fusion peptide and a nuclear localization sequence, are described. A beta-sheet domain is observed in which fluorescence measurements show that ten peptide molecules bind one all-trans retinol or all-trans retinoic acid molecule with a strong affinity (K'd = 40 nM). Stoichiometry and affinity of the binding can be compared with those of cellular retinoid binding proteins, the structure of which is an anti-parallel beta barrel. Analogy between the system under study and cellular retinoid-binding proteins is discussed. Peptide-helped internalization and subsequent perinuclear localization of retinol in human fibroblast cells confirm this analogy. Also, this last result shows that the peptide is an efficient carrier for insoluble substances like retinoids.

Hydrolysis of the major structural polysaccharides of plant cell walls by the aerobic soil bacterium Pseudomonas fluorescens subsp. cellulosa is attributable to the production of multiple extracellular cellulase and hemicellulase enzymes, which are the products of distinct genes belonging to multigene families. Cloning and sequencing of individual genes, coupled with gene sectioning and functional analysis of the encoded proteins have provided a detailed picture of structure/function relationships and have established the cellulase-hemicellulase system of P. fluorescens subsp. cellulosa as a model for the plant cell wall degrading enzyme systems of aerobic cellulolytic bacteria. Cellulose- and xylan-degrading enzymes produced by the pseudomonad are typically modular in structure and contain catalytic and noncatalytic domains joined together by serine-rich linker sequences. The cellulases include a cellodextrinase; a beta-glucan glucohydrolase and multiple endoglucanases, containing catalytic domains belonging to glycosyl hydrolase families 5, 9, and 45; and cellulose-binding domains of families II and X, both of which are present in each enzyme. Endo-acting xylanases, with catalytic domains belonging to families 10 and 11, and accessory xylan-degrading enzymes produced by P. fluorescens subsp. cellulosa contain cellulose-binding domains of families II, X, and XI, which act by promoting close contact between the catalytic domain of the enzyme and its target substrate. A domain homologous with NodB from rhizobia, present in one xylanase, functions as a deacetylase. Mananase, arabinanase, and galactanase produced by the pseudomonad are single domain enzymes. Crystallographic studies, coupled with detailed kinetic analysis of mutant forms of the enzyme in which key residues have been altered by site-directed mutagenesis, have shown that xylanase A (family 10) has 8-fold alpha/beta barrel architecture, an extended substrate-binding cleft containing at least six xylose-binding pockets and a calcium-binding site that protects the enzyme from thermal inactivation, thermal unfolding, and attack by proteinases. Kinetic studies of mutant and wild-type forms of a mannanase and a galactanase from P. fluorescens subsp. cellulosa have enabled the catalytic mechanisms and key catalytic residues of these enzymes to be identified.

Fluorescent proteins (FPs) are ubiquitous in biophysics. To simultaneously tag many different biomolecules and allow excitation at lower wavelengths, FP's have been developed with excitation and fluorescence at wavelengths > 550 nm, however these red fluorescent proteins are more susceptible to photobleaching [1].View Large Image | View Hi-Res Image | Download PowerPoint SlideA possible reason for this is a decrease in the structural stability of the beta barrel, but X-ray B-factor measurements do not indicate this stability change. Zaccai introduced a measure of protein stability called resiliency [2], derived from the temperature dependent atomic mean squared displacement measured by neutron scattering. These facility-based measurements require ∼100 mg of protein, so are not conducive to systematic testing of changes in resiliency with mutation. However table top THz optical absorption measurements have shown the same temperature dependence as [3]. Using this method we find the resiliency of mCherry is 2.5 times higher than mOrange2 consistent with mCherry's higher stability and longer irreversible photobleaching time constant (3.46 s) compared to mOrange (0.26 s).[1] K. M. Dean et al., Biophys. J. 101, 961 (2011).[2] G. Zaccai, Science 288, 1604 (2000).[3] Y. He et al., Phys. Rev. Lett. 101, 178103 (2008).

Elucidating protein plasticity and protein-lipid interactions is critical for a detailed understanding of the workings of membrane protein machines. We study the 88 kDa membrane-embedded protein BamA, which is the main component of the beta-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in gram-negative bacteria. Using a combination of electron microscopy (EM), limited proteolysis and NMR we characterized the fold and dynamics of different BamA constructs and the role of the lipid bilayer for protein structure.Our NMR studies provide atomic insight into protein dynamics and plasticity of the periplasmic POTRA domains and their interactions with the transmembrane domain. Moreover, we studied the influence of lipid type and the lipid-to-protein ratio on the atomic (solid-state NMR) and nm (EM) scale. Taken together, our results provide first clues into the interplay between protein - protein and protein -lipid interactions that may be critical for BAM-mediated substrate insertion into the outer membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide

In order to help determine the extent to which side chain interactions within the staphylococcal nuclease beta-barrel affect its global stability, a full set of point mutants was generated for residue 27. Intrinsic tryptophan fluorescence was monitored during solvent denaturation with guanidine hydrochloride (GuHCl) and was used to calculate DeltaGH2O unfolding and m values for each mutant. In the wild type protein, residue 27 is a tyrosine which is at the first position of a type I' beta-turn, and which participates in both hydrophobic interactions and side chain to side chain hydrogen bonding. The hydrophobicity of the mutant residue was found to be the dominant factor in determining global protein stability within this series of nuclease mutants.

The mechanical response of a TIM-barrel protein to an applied pressure has been studied. We generated structures under an applied pressure by assuming the volume change to be a linear function of normal mode variables. By Delaunay tessellation, the space occupied by protein atoms is divided uniquely into tetrahedra, whose four vertices correspond to atomic positions. Based on the atoms that define them, the resulting Delaunay tetrahedra are classified as belonging to various secondary structures in the protein. The compressibility of various regions identified with respect to secondary structural elements in this protein is obtained from volume changes of respective regions in two structures with and without an applied pressure. We found that the beta barrel region located at the core of the protein is quite soft. The interior of the beta barrel, occupied by side chains of beta strands, is the softest. The helix, strand, and loop segments themselves are extremely rigid, while the regions existing between these secondary structural elements are soft. These results suggest that the regions between secondary structural elements play an important role in protein dynamics. Another aspect of tetrahedra, referred to as bond distance, is introduced to account for rigidities of the tetrahedra. Bond distance is a measure of separation of the atoms of a tetrahedron in terms of number of bonds along the polypeptide chain or side chains. Tetrahedra with longer bond distances are found to be softer on average. From this behavior, we derive a simple empirical equation, which well describes the compressibilities of various regions.

The large subunit of ribonucleotide reductase (RNR) contains a ten-stranded beta/alpha barrel of a new type consisting of two antiparallel halves. The two halves of the barrel are pseudo 2-fold-related, have similar folds but different additional intervening secondary structure elements and loops. The inner diameter of the RNR barrel, 15 A to 20 A, is significantly larger than for the (beta alpha)3 barrels. The larger barrel forms a stable framework which holds an inserted hairpin loop rigidly and exposes active site residues at its tip. The barrel organization allows three cysteine residues to be positioned close to each other without forming unfavorable disulfide bridges between Cys439 on the tip of the inserted loop and the redox-active cysteine residues on the barrel strands. Redox-active cysteine residues separated by more than 200 residues are held in close proximity to each other on adjacent barrel strands.

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

Triosephosphate isomerase (TIM) of the hyperthermophilic Archaea Pyrococcus woesei and Methanothermus fervidus have been purified to homogeneity. The enzymes from the two hyperthermophiles represent homo-tetramers of 100 kDa, contrary to all known bacterial and eukaryotic TIMs, which are dimers of 48–60 kDa. Molecular size determination of the TIM from the mesophilic methanogen Methanobacterium bryantii yielded the usual molecular mass of only 57 kDa, indicating that the tetrameric aggregation state does not represent an archaeal feature but rather correlates with thermoadaptation. A similar preference for higher protein aggregates in hyperthermophilic Archaea has previously been demonstrated for 3-phosphoglycerate kinases. The gene of the P. woesei TIM was cloned and sequenced. The archaeal TIM proved to be homologous to its bacterial and eukaryotic pendants. Most strikingly, the deduced protein sequence comprises only 224 residues and thus represents the shortest TIM sequence known as yet. Taking the three-dimensional structure of the eucaryal TIM as a basis, from the shortenings of the chain considerable rearrangements at the bottom of the α/β barrel and at its functionally inactive flank are expected, which are interpreted in terms of the formation of new subunit contacts.

Fructuose bisphostate aldolase (FBA) and triosephosphate isomerase (TIM) are the fourth and fifth enzymes in the glycolysis pathway and they are known to bind. FBA cleaves the six-carbon fructose 1, 6-bisphosphate into two three-carbon components – dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). TIM converts DHAP into GAP, a substrate for the subsequent synthetic step. These two alpha/beta barrel proteins have high structural similarity – with a core RMSD 4.8A. These two enzymes have low activity in the monomeric form with the functionally active structures present in higher oligomeric states. By applying Elastic Network Model, we find the modes of motions that are functionally important for these proteins. We build models for the complex between these two proteins to investigate their important motions in their complexes as well as for their different oligomeric states including those that are different in different species to learn their important modes of motions for different functionalities. For each protein, by multiple sequence alignment across the species, we predict the coevolving residues and cluster these residues along the structure. We build the information transfer pathways from the important interface residues to the catalytic residues. Change in these pathways in different oligomeric states maybe be related to the change of motions in the catalytic region in different oligomeric states. We use this knowledge about the changes in motions and the information transfer pathways within the structure of these interacting proteins as constraints for selecting the computational docked models of complexes between these two proteins to preserve their functional motions.

We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an R-factor of 16.8%. An enzyme from the beta-ketoadipate pathway, MLE catalyses the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one monomer consisting of 373 amino acid residues. MLE has two large domains and a C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a C-terminal meandering subdomain. The alpha beta-barrel domain is highly irregular. Its structure is (beta/alpha)7 beta, with the structural role of the last alpha-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain. The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes. The strand crossing angles also vary enormously, from +9 degrees to -69 degrees; the first and last strands, which close the barrel, cross at an angle of -69 degrees, making extensive strand-strand hydrogen bonding impossible. The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other alpha beta-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel beta-strands. The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules. The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions. Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior. The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively. This might account for the irregularity of the barrel. The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO- of muconolactone superimposed on the corresponding atoms of cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product.(ABSTRACT TRUNCATED AT 400 WORDS)

The structure of NADH-cytochrome b5 reductase from pig liver microsomes has been refined to a crystallographic R factor of 0.223 at 2.4 Å resolution. A structural comparison between the flavin-binding β barrel domain of NADH-cytochrome b5 reductase and those of the other flavin-dependent reductases, ferredoxin-NADP+ reductase, phthalate dioxygenase reductase and nitrate reductase, indicated that the overall barrel foldings are similar to each other and that the specific arrangement of three amino acid residues (Arg, Tyr and Ser/Thr) is usually necessary for flavin-binding. These conserved residues overlap each other in their three-dimensional structures and stabilize the flavin-binding site in the four flavin-dependent reductases.

The solution structure of the antifungal protein (AFP, 51 residues, 4 disulfide bridges) from Aspergillus giganteus has been determined by using experimentally derived interproton distance constraints from nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific proton assignments were obtained at pH 5.0 and 35 °C. A set of 834 upper limit distance constraints from nuclear Overhauser effect measurements was used as input for the calculation of structures with the program DIANA. An initial family of 40 structures calculated with no disulfide constraints was used to obtain information about the disulfide connectivities, which could not be determined by standard biochemical methods. Three possible disulfide patterns were selected and the corresponding disulfide constraints applied to generate a family of 20 DIANA conformers for each pattern. Following energy minimization, the average pairwise RMSD of the 20 conformers of each family is 1.01,0.89, and 1.01 Å for backbone atoms and 1.82, 1.74, and 1.81 Å for all heavy atoms. One of these three families contains the disulfide bridge arrangement actually present in the solution structure of AFP. Although the three families fulfill the NMR constraints, one of the disulfide patterns considered (cysteine pairs 7-33, 14-40, 26-49, 28-51) is favored among the others on the basis of previous chemical studies. It thus probably corresponds to the actual pattern of disulfide bridges present in the protein, and the corresponding family represents the solution structure of AFP. The folding of AFP consists of five antiparallel β strands connected in a -1, - 1, +3, +1 topology and highly twisted, defining a small and compact β barrel stabilized by four internal disulfide bridges. A cationic site formed by up to three lysine side chains adjacent to a hydrophobic stretch, both at the protein surface, may constitute a potential binding site for phospholipids which would be the basis of its biological function. On the other hand, a second, minor form of AFP has been detected. NMR data, together with results from mass spectrometry, chemical analysis, and sedimentation equilibrium, suggest that this species differs from the major form in the pairs of cysteines involved in the four disulfide bridges., This work was supported by the Dirección General de Investigación Científica y Técnica (Spain) (PB90/0120 and PB93/0090). R.C.O. was recipient of a predoctoral grant (FP92-02882770) from the Ministerio de Educación y Ciencia (Spain).

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or “sequence conservation” as the basis for their understanding. Recently “interaction energy” based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the “interaction conservation” viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design., Author Summary Proteins are polymers of amino-acids that fold into unique three-dimensional structures to perform cellular functions. This structure formation has been shown to depend on the amino-acid sequences. But examples of proteins with diverse sequences retaining a similar structural fold are quite substantial that we can no longer consider such phenomenon as exceptions. Therefore, this non-canonical relationship has been studied extensively mostly by studying the remote sequence similarities between proteins. Here we have attempted to address the above-mentioned problem by analyzing the similarities in the spatial interactions among amino-acids. Since the protein structure is a resultant of different interactions, we have considered the proteins as networks of interacting amino-acids to derive the common interactions within a popular structural fold called the TIM barrel fold. We were able to find common interactions among different families of the TIM fold and generalize the patterns of interactions by which the fold is being maintained despite sequence diversity. The results substantiate our hypothesis that interaction conservation might by a driving factor in fold formation and this new outlook can be used extensively in engineering proteins with better biophysical characteristics.

Optimizing protein expression is a key step for NMR spectroscopy, which typically requires milligram quantities of isotope-labeled protein.Human cardiac troponin I (cTnI) is a 210-amino acid protein that accumulates in intracellular inclusion bodies when overexpressed in E. coli. Our initial attempts at producing an isolated N-terminal fragment, cTnI[1-71], resulted in negligible expression. This was due to proteolytic degradation, as evidenced by the expression of a fusion construct, GB1-cTnI[1-71], yielding GB1 alone. In order to target cTnI[1-71] to inclusion bodies, we fused it to Comamonas testosteroni ketosteroid isomerase (KSI), a hydrophobic 125-amino acid protein, using the commercially available pET31b expression vector.The KSI-cTnI[1-71] fusion protein was expressed in inclusion bodies and purified to a reasonable yield. However, when a tandem dimer, KSI-cTnI[1-71]-cTnI[1-71], was expressed, only a single band corresponding to the molecular weight of KSI alone was observed, suggesting that it was susceptible to intracellular proteases.Beta barrel proteins of the Gram negative bacterial outer membrane accumulate in inclusion bodies when overexpressed without their N-terminal signal sequence. A fusion construct involving the E. coli outer membrane enzyme, PagP, (PagP-cTnI[1-71]) expressed robustly in inclusion bodies. Moreover, the tandem dimer, PagP-cTnI[1-71]-cTnI[1-71], also expressed well.Using a beta barrel protein as a fusion partner has several theoretical advantages for expressing intrinsically disordered proteins. There is no possibility for the protein to fold outside of the membrane environment. Furthermore, beta barrel membrane proteins are not extremely hydrophobic, since the interior of the barrel is typically hydrophilic. Hydrophobic proteins tend to have limited yield, can be toxic to cells, and may interfere with later purification or processing steps. Thus fusion to PagP or other beta barrel membrane proteins represents a promising new alternative for difficult-to-express disordered proteins.

Aptamers are specific oligonucleotide molecules that bind to various biologically important target molecules. Nanopores are powerful tools for the identification and detection of single molecules, such as DNA, RNA and proteins. α-hemolysin nanopore is utilized to sense the single protein molecule with a transfer-aptamer (piston). The transfer-aptamer (piston) containing polyethylene glycol (PEG) has functions as identified which zone of the beta barrel of pore. Here we report the formation of reversible signature signals using protein engineering, and characterized change the pore conductance depend on special design of transfer-aptamer (piston). The transfer-aptamer was bound with thrombin, which can be trapped to the pore opening and distinguished signature current block compared the thrombin being released from the transfer-aptamer (piston). But, the signal of aptamer only contains DNA which is indistinguishable with or without thrombin. By containing specific tail of aptamer, presence of single protein molecule, we capture the reversible thrombin signature signal using protein pore. The transfer-aptamer (piston) can be used as detector of different ions for nanomachine, nanomaterials, and folding process.

Organophosphates, such as parathion and paraoxon, constitute the largest class of insecticides currently used in industrialized nations. In addition, many of these compounds are known to inhibit mammalian acetylcholinesterases thereby acting as nerve agents. Consequently, organophosphate-degrading enzymes are of considerable interest in light of their ability to detoxify such compounds. Here we report the three-dimensional structure of such an enzyme, namely, phosphotriesterase, as determined by single crystal X-ray diffraction analysis to 2.1-A resolution. Crystals employed in this investigation belonged to the space group P2(1)2(1)2 with unit cell dimensions of a = 80.3 A, b = 93.4 A, and c = 44.8 A and one molecule per asymmetric unit. The structure was solved by multiple isomorphous replacement with two heavy-atom derivatives and refined to a crystallographic R factor of 18.0%. As observed in various other enzymes, the overall fold of the molecule consists of an alpha/beta barrel with eight strands of parallel beta-pleated sheet. In addition, there are two antiparallel beta-strands at the N-terminus. The molecular model of phosphotriesterase presented here provides the initial structural framework necessary toward understanding the enzyme's broad substrate specificities and its catalytic mechanism.

In a rational protein design approach, the His3 Zn(II)-binding site from the active center of human carbonic anhydrase II was transplanted on the beta-barrel of mammalian serum retinol-binding protein (RBP) in a solvent-accessible location on the protein's outer surface. Several mutants of RBP were generated and produced in Escherichia coli, and their Zn(II)-binding properties were investigated in equilibrium dialysis experiments. One mutant, RBP/H3(A), with His residues introduced at the positions 46, 54, and 56 in the polypeptide sequence was shown to bind Zn(II) specifically with a stoichiometry of 1 and a corresponding dissociation constant equal to 36 +/- 10 nM. Binding of Zn(II) had no influence on the binding of retinoic acid, a natural ligand of RBP. In guanidinium chloride-induced unfolding experiments the mutant was found to be significantly stabilized in the presence of small concentrations of ZnSO4. This effect could be quantitatively explained using thermodynamic theory. Furthermore, it was demonstrated that the protein-bound Zn(II) is accessible to iminodiacetic acid as an additional chelating ligand without competition for the metal ion. Thus it was possible to use the grafted metal-binding site for the efficient purification of the engineered, bifunctional RBP via immobilized metal affinity chromatography from the bacterial protein extract.

Alpha/beta barrels have an ill-defined origin. Evidence exists which favours their divergent evolution from a common ancestral barrel and convergent evolution to a stable fold. However, recent sequence and structural information for the flavin oxidase/dehydrogenase family of barrel enzymes indicate that sub-families of alpha/beta barrels have evolved divergently. The modular fusion of barrel domains with core structures from other gene families has also contributed to the evolution of related but catalytically distinct enzyme molecules within each sub-family of the flavin oxidases/dehydrogenases. An analysis of the structures and sequences of the flavin oxidases/dehydrogenases has now enabled studies focusing on the evolutionary origins and modular assembly of this important family of proteins to be initiated.

The vast majority of currently structurally characterised outer membranea proteins of E.coli are beta barrel porins that exist either as monomers or homo-oligomers. The function of these porins is varied and complex and is thought to depend not just on their structural dynamics but also on interactions both with other porins and with their lipid environment.To provide an insight into these processes we study in detail the motion and arrangement of both lipids and proteins within a series of model systems.We represent the outer membrane as a bilayer consisting of mixtures of phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) lipids and examine by means of atomistic and coarse grained simulations the relative dynamics of a representative sample of outer membrane proteins. We consider the diffusive ability of both isolated, and multiple proteins within larger systems which more realistically reflects the density of porins within the outer membrane. The diffusive behaviour of proteins is related to protein characteristics such as size and localised bilayer deformation.

The cell cycle regulatory protein CksHs2 binds to the catalytic subunit of the cyclin-dependent kinases (Cdk9s) and is essential for their biological function. The crystal structure of the protein was determined at 2.1 A resolution. The CksHs2 structure is an unexpected hexamer formed by the symmetric assembly of three interlocked dimers into an unusual 12-stranded beta barrel fold that may represent a prototype for this class of protein structures. Sequence-conserved regions form the unusual beta strand exchange between the subunits of the dimer, and the metal and anion binding sites associated with the hexamer assembly. The two other sequence-conserved regions line a 12 A diameter tunnel through the beta barrel and form the six exposed, charged helix pairs. Six kinase subunits can be modeled to bind the assembled hexamer without collision, and therefore this CksHs2 hexamer may participate in cell cycle control by acting as the hub for Cdk multimerization in vivo.

To understand the structure of the hormone binding domain (HBD) of human beta 1 thyroid hormone nuclear receptor (h-TR beta 1), truncated h-TR beta 1 fragments, MD32 (M169-D456), KD29 (K201-D456), DD28 (D211-D456), KD25 (K235-D456), and KP28 (K201-P448), were analyzed by circular dichorism (CD). MD32 and KD29 show intense CD spectra with double minima at 222 and 208-210 nm, indicating the presence of extensive regions of alpha-helix. DD28 and KD25 have spectra which are reduced in intensity with minima around 215 nm, characteristic of a beta-sheet. The observed spectra are compatible with sequence analysis which predicts that HBD contains alternating stretches of alpha-helix and beta-strand. These extensive decreases in secondary structure in DD28 and KP28 in which the predicted first beta-strand or last alpha-helix was deleted, respectively, were accompanied by the loss of hormone binding activity. On the basis of these results, we suggest a new model for h-TR beta 1 consisting of the known DNA binding domain linked by an alpha-helical hinge to the HBD, with the tertiary structure of an alpha/beta barrel. The model is compatible with previous chemical and genetic studies on the structure of this protein.

Rapid diffusion of hydrophilic molecules across the outer membrane of mitochondria has been related to the presence of a protein of 29 to 37 kDa, called voltage-dependent anion channel (VDAC), able to generate large aqueous pores when integrated in planar lipid bilayers. Functional properties of VDAC from different origins appear highly conserved in artificial membranes: at low transmembrane potentials, the channel is in a highly conducting state, but a raise of the potential (both positive and negative) reduces drastically the current and changes the ionic selectivity from slightly anionic to cationic. It has thus been suggested that VDAC is not a mere molecular sieve but that it may control mitochondrial physiology by restricting the access of metabolites of different valence in response to voltage and/or by interacting with a soluble protein of the intermembrane space. The latest application of the patch clamp and tip-dip techniques, however, has indicated both a different electric behavior of the outer membrane and that other proteins may play a role in the permeation of molecules. Biochemical studies, use of site-directed mutants, and electron microscopy of two-dimensional crystal arrays of VDAC have contributed to propose a monomeric beta barrel as the structural model of the channel. An important insight into the physiology of the inner membrane of mammalian mitochondria has come from the direct observation of the membrane with the patch clamp. A slightly anionic, voltage-dependent conductance of 107 pS and one of 9.7 pS, K(+)-selective and ATP-sensitive, are the best characterized at the single channel level. Under certain conditions, however, the inner membrane can also show unselective nS peak transitions, possibly arising from a cooperative assembly of multiple substrates.

The denaturation of prion protein (PrP) and the concurrent formation of beta-sheet rich isoform has been accredited to the etiology of the prion diseases. Accumulating biochemical evidences underline the critical role of oligomeric PrP conformations emerging from the early stage of the denaturation process. However detailed structural information on the oligomeric isoforms remains elusive, which hampers precise description of the pathological process. Recently we proposed a new structural hypothesis for the oligomeric PrP species comprised of a short PrP construct (Human PrP 175-217) based on experimental findings. We postulated that 1) monomers adopt beta-hairpin conformation and 2) assemble as beta-barrel quaternary structure. These assumptions provided a comprehensive explanation for the experimental findings suggesting beta-sheet rich structure including circular dichroism (CD) spectrum and Fourier transformed infra-red spectroscopy (FTIR) as well as the presence of disulfide bridge between CYS-179 and CYS-214. To be more specific, we constructed various beta-barrel models differing in number of monomers, inter-molecular hydrogen bond pattern and side-chains facing exterior of the barrel. Those models were refined extensively using a protein structure prediction tool (Rosetta). Structural energy profile of the predicted oligomer models was consistently lower than that of native like monomer or oligomers comprised of partially denatured monomers. Also the smallest stable oligomer was predicted to be an octamer, which is in good agreement with available mass spectrometric data. Finally, we discussed a possible generality between protein denaturation and amyloidogenesis problems in general, by comparing our model with a oligomeric assembly model for the pathological amyloid beta protein (Abeta).

The extracellular hemoglobin of Ascaris has an extremely high oxygen affinity (P50 = 0.004 mmHg). It consists of eight identical subunits of molecular weight 40,600. Their sequence, determined by protein chemistry, shows two tandemly linked globin-like sequences and an 18-residue C-terminal extension. Two N-linked glycosylation sites contain equal ratios of mannose/glucosamine/fucose of 3:2:1. Electron micrographs suggest that the eight subunits form a polyhedron of point symmetry D4, or 42. The C-terminal extension contains a repeat of the sequence Glu-Glu-His-Lys, which would form a pattern of alternate glutamate and histidine side chains on one side and of glutamate and lysine side chains on the other side of a beta strand. We propose that this represents a polar zipper sequence and that the C-terminal extensions are joined in an eight-stranded beta barrel at the center of the molecule, with histidine and glutamate side chains inside and lysine and glutamate side chains outside the barrel compensating each other's charges. The amino acid sequence of Ascaris hemoglobin fails to explain its high oxygen affinity.

Background A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA) moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2beta2-2beta3 loops of the heptameric precursor to generate a 14-strand transmembrane beta barrel. Methodology/principal findings We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the beta barrel, and a third one, F324, that lies part way up the barrel. Conclusions/significance Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. We have doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins share a transmembrane β-barrel motif, but vary in barrel size and primary sequence. We quantified their ability to fold into a matrix of bilayer environments by SDS-PAGE. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. We show that while environmental factors are influential, OMP folding must also be modulated by intrinsic protein properties. To rationalize the differences in folding results between OMPs, we explored their kinetic profile and determined their resistance to thermal denaturation. Although these proteins share a common structural motif and have evolved to reside in the same bilayer environment in vivo, we found that these OMPs vary in their folding behavior.

Many data suggest that specific sequence characteristics affect the ability to form amyloid fibrils. To pinpoint that structural variation in amino acids are responsible for transforming normal soluble proteins to insoluble amyloid aggregates, we propose that this process is similar to the interconversion of two types of beta proteins – sandwich proteins (SP) and barrel proteins (BP). We determine and compare conserved residues, which are common to SP and BP, which we call structural determinants of SP and BP. Common methods of sequence alignment for identification of conserved residues cannot be applied here because analysed sequences are very diverse. To uncover structural determinants, a new method of structural-based sequence alignment was applied, which allows comparing sequences with very low sequence similarity, but similar 3D structures. For analysis and comparison of structural determinants of BP and SP, a recently suggested scale of hydrophobicity was used. It allowed us to reveal delicate sequence features, which distinguish SP and BP and are likely responsible for amyloid aggregation.

We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein Engng, 3, 259-266]. Infrared and Raman spectra analyses of octarellin's secondary structure reveal the expected percentage of alpha-helices (30%) and a higher beta-sheet content (40%) than predicted from the design. When the Raman spectra obtained with octarellin and native triosephosphate isomerase (a natural alpha/beta-barrel) are compared, similar percentages of secondary structures are found. Thermal denaturation of octarellin monitored by CD confirms that its secondary structures are quite stable, whereas its native-like tertiary fold is not. Tyrosine residues, predicted to be partially hidden from solvent, are actually exposed as revealed by Raman and UV absorption spectra. We conclude that the attempted alpha/beta-barrel conformation in octarellin may be loosely packed. The criteria used to design octarellin are discussed and improvements suggested.

The crystal structure of a murine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, has been determined and refined at 2.4 angstrom resolution. The structure is folded as an eight-stranded parallel alpha/beta barrel with a deep pocket at the beta-barrel COOH-terminal end wherein the inhibitor and a zinc are bound and completely sequestered. The presence of the zinc cofactor and the precise structure of the bound analog were not previously known. The 6R isomer of the analog is very tightly held in place by the coordination of the 6-hydroxyl to the zinc and the formation of nine hydrogen bonds. On the basis of the structure of the complex a stereoselective addition-elimination or SN2 mechanism of the enzyme is proposed with the zinc atom and the Glu and Asp residues playing key roles. A molecular explanation of a hereditary disease caused by several point mutations of an enzyme is also presented.

The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel beta barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (A) long protrusion on the threefold axes, a 15 A deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 A deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the alpha subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding. Previous studies [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965-2974] have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units. In terms of the secondary structure, the amino folding unit consists of the first six strands and five alpha helices of this alpha/beta barrel protein. The carboxy folding unit comprises the remaining two strands and three alpha helices; position 211 is in strand 7. Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly. In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation. The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles. When considered with previous data on replacements at other positions in the alpha subunit [Hurle, M. R., Tweedy, N. B., & Matthews, C. R. (1986) Biochemistry 25, 6356-6360], it is clear that beta strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process.

The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

Rv0813c is a protein of unknown function that we have selected as a target for crystallographic studies in the context of a structural genomics effort on tuberculosis. The crystal structure of Rv0813c, a conserved protein in M. tuberculosis, reveals a new family of putative fatty acid binding proteins (FABPs). Rv0813c adopts a 10-stranded beta barrel fold, which closely resembles those of the FABPs found in eukaryotes. This is in fact the first FABP-like protein to be found in prokaryotes. However, Rv0813c lacks the double helix insert of FABPs that covers the entry to the binding site. The beta barrel forms a deep cavity, where a small ligand, which appears to be a morpholine, binds to the phenol hydroxyl group of Tyr192. This tyrosine corresponds to a RxY motif, which forms part of the binding site in FABPs. Furthermore, a network of H bonds, hydrophobic residues and an internal salt bridge surround the binding site and define the shape of the cavity. Most of these residues are well conserved in homologous proteins. Phylogenetic studies show that this family of FABP-like proteins is represented in GC-rich prokaryotes. The structural analysis of Rv0813c suggests that this cytoplasmic protein may have a role in fatty acid transport, storage or signaling. This work supports the notion that high resolution structural studies can provide strong leads as to the biochemical function(s) of the protein.

Background Beta-barrel transmembrane (bbtm) proteins are a functionally important and diverse group of proteins expressed in the outer membranes of bacteria (both gram negative and acid fast gram positive), mitochondria and chloroplasts. Despite recent publications describing reasonable levels of accuracy for discriminating between bbtm proteins and other proteins, screening of entire genomes remains troublesome as these molecules only constitute a small fraction of the sequences screened. Therefore, novel methods are still required capable of detecting new families of bbtm protein in diverse genomes.

Nature 403, 617–622 (2000). This laboratory reported the in vitro evolution of an enzyme with phosphoribosyl anthranilate isomerase activity (ivePRAI) from the α/β-barrel scaffold of indole-3-glycerol-phosphate synthase using a combination of rationally designed libraries, DNA shuffling, and selection with Escherichia coli, JA300, a strain that lacks an active PRAI gene.