Showing papers in "Protein Science in 2001"

Sequence-structure analysis of FAD-containing proteins.

Abstract: We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein `pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.

Molecular confinement influences protein structure and enhances thermal protein stability.

Abstract: The sol-gel method of encapsulating proteins in a silica matrix was investigated as a potential experimental system for testing the effects of molecular confinement on the structure and stability of proteins. We demonstrate that silica entrapment (1) is fully compatible with structure analysis by circular dichroism, (2) allows conformational studies in contact with solvents that would otherwise promote aggregation in solution, and (3) generally enhances thermal protein stability. Lysozyme, α-lactalbumin, and metmyoglobin retained native-like solution structures following sol-gel encapsulation, but apomyoglobin was found to be largely unfolded within the silica matrix under control buffer conditions. The secondary structure of encapsulated apomyoglobin was unaltered by changes in pH and ionic strength of KCl. Intriguingly, the addition of other neutral salts resulted in an increase in the α-helical content of encapsulated apomyoglobin in accordance with the Hofmeister ion series. We hypothesize that protein conformation is influenced directly by the properties of confined water in the pores of the silica. Further work is needed to differentiate the steric effects of the silica matrix from the solvent effects of confined water on protein structure and to determine the extent to which this experimental system mimics the effects of crowding and confinement on the function of macromolecules in vivo.

Entropy—enthalpy compensation: Fact or artifact?

Abstract: The phenomenon of entropy–enthalpy (S-H) compensation is widely invoked as an explanatory principle in thermodynamic analyses of proteins, ligands, and nucleic acids. It has been suggested that this compensation is an intrinsic property of either complex, fluctuating, or aqueous systems. The questions examined here are whether the observed compensation is extra-thermodynamic (i.e., reflects anything more than the well-known laws of statistical thermodynamics) and if so, what does it reveal about the system? Compensation is rather variably defined in the literature and different usages are discussed. The most precise and interesting one, which is considered here, is a linear relationship between ΔH and ΔS for some series of perturbations or changes in experimental variable. Some recent thermodynamic data on proteins purporting to show compensation is analyzed and shown to be better explained by other causes. A general statistical mechanical model of a complex system is analyzed to explore whether and under what conditions extra-thermodynamic compensation can occur and what it reveals about the system. This model shows that the most likely behavior to be seen is linear S-H compensation over a rather limited range of perturbations with a compensation temperature Tc = dΔH/dΔS within 20% of the experimental temperature. This behavior is insensitive to the details of the model, thus revealing little extra-thermodynamic or causal information about the system. In addition, it will likely be difficult to distinguish this from more trivial forms of compensation in real experimental systems.

Black hole entropy, special geometry and strings

Abstract: We review work done over the last years on the macroscopic and microscopic entropy of supersymmetric black holes in fourdimensional = 2 supergravity and in = 2 compactifications of string theory and M-theory. Particular emphasis is put on the crucial role of higher curvature terms and of modifications of the area law in obtaining agreement between the macroscopic entropy, which is a geometric property of black hole solutions and the microscopic entropy, which is computed by state counting in Calabi-Yau compactifications of string or M-theory. We also discuss invariance properties of the entropy under stringy T-duality and S-duality transformations in = 2,4 compactifications in presence of higher curvature terms. In order to make the paper self-contained we review the laws of black hole mechanics in higher derivative gravity, the definition of entropy as a surface charge, the superconformal off-shell description of = 2 supergravity, special geometry, and = 2 compactifications of heterotic and type II string theory and of M-theory.

Comparing function and structure between entire proteomes

Abstract: More than 30 organisms have been sequenced entirely. Here, we applied a variety of simple bioinformatics tools to analyze 29 proteomes for representatives from all three kingdoms: eukaryotes, prokaryotes, and archaebacteria. We confirmed that eukaryotes have relatively more long proteins than prokaryotes and archaes, and that the overall amino acid composition is similar among the three. We predicted that ∼15%–30% of all proteins contained transmembrane helices. We could not find a correlation between the content of membrane proteins and the complexity of the organism. In particular, we did not find significantly higher percentages of helical membrane proteins in eukaryotes than in prokaryotes or archae. However, we found more proteins with seven transmembrane helices in eukaryotes and more with six and 12 transmembrane helices in prokaryotes. We found twice as many coiled-coil proteins in eukaryotes (10%) as in prokaryotes and archaes (4%–5%), and we predicted ∼15%–25% of all proteins to be secreted by most eukaryotes and prokaryotes. Every tenth protein had no known homolog in current databases, and 30%–40% of the proteins fell into structural families with >100 members. A classification by cellular function verified that eukaryotes have a higher proportion of proteins for communication with the environment. Finally, we found at least one homolog of experimentally known structure for ∼20%–45% of all proteins; the regions with structural homology covered 20%–30% of all residues. These numbers may or may not suggest that there are 1200–2600 folds in the universe of protein structures. All predictions are available at http://cubic.bioc.columbia.edu/genomes.

Strain-specified relative conformational stability of the scrapie prion protein.

Abstract: Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrPSc). To explore this postulate, we examined the properties of PrPSc from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrPSc molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrPSc as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have ∼75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a ∼170-d incubation period and ∼1.25 M for the SHa(RML) and 139H isolates with ∼180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a ∼320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrPSc conformers encipher the biological properties of prion strains.

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

Abstract: The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

A structural basis for processivity

Abstract: The structures of a number of processive enzymes have been determined recently. These proteins remain attached to their polymeric substrates and may perform thousands of rounds of catalysis before dissociating. Based on the degree of enclosure of the substrate, the structures fall into two broad categories. In one group, the substrate is partially enclosed, while in the other class, enclosure is complete. In the latter case, enclosure is achieved by way of an asymmetric structure for some enzymes while others use a symmetrical toroid. In those cases where the protein completely encloses its polymeric substrate, the two are topologically linked and an immediate explanation for processivity is provided. In cases where there is only partial enclosure, the structural basis for processivity is less obvious. There are, for example, pairs of proteins that have quite similar structures but differ substantially in their processivity. It does appear, however, that the enzymes that are processive tend to be those that more completely enclose their substrates. In general terms, proteins that do not use topological restraint appear to achieve processivity by using a large interaction surface. This allows the enzyme to bind with moderate affinity at a multitude of adjacent sites distributed along its polymeric substrate. At the same time, the use of a large interaction surface minimizes the possibility that the enzyme might bind at a small number of sites with much higher affinity, which would interfere with sliding. Proteins that can both slide along a polymeric substrate, and, as well, recognize highly specific sites (e.g., some site-specific DNA-binding proteins) appear to undergo a conformational change between the cognate and noncognate-binding modes.

Solubilization and disaggregation of polyglutamine peptides

Abstract: A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q20 have been reported to be insoluble in water, but dissolution in--and evaporation from--a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q44 to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies-including in vitro aggregation experiments--on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.

A refined solution structure of hen lysozyme determined using residual dipolar coupling data.

Abstract: A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H–15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low-energy calculated structures has an average backbone RMSD of 0.50±0.13Å to the mean structure and of 1.49±0.10Å to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation-dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.

Crystal structure of an archaeal DNA sliding clamp: Proliferating cell nuclear antigen from Pyrococcus furiosus

Abstract: DNA replication is a fundamental event for the maintenance of life. The molecular apparatus acting in this process has been well characterized in Bacteria and Eucarya. The proteins with common roles in the DNA replication mechanism have been identified from these two biological domains, even though the amino acid sequences between the proteins from Bacteria and Eucarya are quite different (Kornberg and Baker 1992; Stillman 1994; Kelman and O'Donnell 1995; Waga and Stillman 1998). The replicative DNA polymerases require an elongation factor, called the sliding clamp, for processive DNA synthesis. Proliferating cell nuclear antigen (PCNA) plays this role in the eukaryotic system. Bacterial DNA polymerase III β subunit (Stukenberg et al. 1991) and bacteriophage T4 gene 45 protein (gp45) (Huang et al. 1981) have long been known to be the sliding clamps for their own DNA polymerases. The amino acid sequences of these sliding clamps are very different from each other. The crystal structures of PCNA (Krishna et al. 1994; Gulbis et al. 1996), β subunit (Kong et al. 1992), gp45 (Moarefi et al. 2000), and bacteriophage RB69 gp43 (Shamoo and Steitz 1999) have been solved, and they all have in common a ring-shaped structure with a pseudo-sixfold symmetry. The homotrimer of PCNA, gp45, and gp43 or the homodimer of β subunit molecules encircles the DNA strand in its central cavity and can slide freely along double-stranded DNA. The sliding clamps directly interact with the replicative DNA polymerases (Polymerase [Pol] δ and Pol ɛ for PCNA, Pol III for β subunit, T4 Pol for gp45, and RB69 Pol for gp43) and enhance their processive DNA synthesis. In addition to its role as the sliding clamp for DNA polymerases, PCNA communicates with a wide variety of proteins involved in the important cellular processes, including cell cycle control, DNA repairs, and an apoptotic pathway (Jonsson and Hubscher 1997; Kelman 1997; Kelman and Hurwitz 1998; Tsurimoto 1998). In an effort to understand the molecular recognition mechanisms between PCNA and these proteins, many studies on the structure–function relationships have been reported to date. Recent genome analyses of Archaea, the third domain of life, showed that the proteins related to genetic information, including DNA replication, are structurally more similar to eukaryotic proteins than to those from Bacteria (Edgell and Doolittle 1997; Ishino and Cann 1998; Cann and Ishino 1999). We cloned a gene with a sequence homologous to eukaryotic PCNA from the hyperthermophilic euryarchaeote Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the purified gene product (Cann et al. 1999). The protein interacted with both DNA polymerase I and II in this organism and enhanced their DNA synthesizing activities; therefore, we named it PfuPCNA. A chemical cross-linking experiment showed that PfuPCNA can also form a trimeric structure. In a characterization of the PCNA homologs in the other archaea, Sulfolobus solfataricus and Methanobacterium thermoautotrophicum, it has been reported that they also stimulate DNA polymerization (De Felice et al. 1999; Kelman and Hurwitz 2000). These results suggest that the basic mechanism for processive DNA synthesis is conserved in the three biological domains. In this study, we crystallized the PfuPCNA and solved its three-dimensional structure. The ring-shaped structure with the pseudo-sixfold symmetry of the sliding clamps was clearly conserved in the archaeal molecule.

MPtopo: A database of membrane protein topology

Abstract: The reliability of the transmembrane (TM) sequence assignments for membrane proteins (MPs) in standard sequence databases is uncertain because the vast majority are based on hydropathy plots. A database of MPs with dependable assignments is necessary for developing new computational tools for the prediction of MP structure. We have therefore created MPtopo, a database of MPs whose topologies have been verified experimentally by means of crystallography, gene fusion, and other methods. Tests using MPtopo strongly validated four existing MP topology-prediction algorithms. MPtopo is freely available over the internet and can be queried by means of an SQL-based search engine.

Structure Determination of Human and Murine Beta-Defensins Reveals Structural Conservation in the Absence of Significant Sequence Similarity

Abstract: Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian β-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human β-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that β-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the β-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because β-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

Some thermodynamic implications for the thermostability of proteins

Abstract: An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water-soluble proteins surveyed by Robertson and Murphy occurs at T* ∼283K, independent of the heat denaturation temperature, T_m. This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of ∼0.008 kJ/mole/residue is, on average, associated with a 1°C increase in T_m. An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins

Abstract: Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.

The Holographic renormalization group

Abstract: In this lecture, we review the derivation of the holographic renormalization group given in hep-th/9912012. Some extra background material is included, and various applications are discussed.

The PYRIN domain: a member of the death domain-fold superfamily.

Abstract: PYRIN domains were identified recently as putative protein–protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The ∼95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein–protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.

Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M.

Abstract: We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g ↔ e′ (i, i′ +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (Tm = 28°C) or nanomolar (Tm = 40°C) range, respectively, but heterodimerize with a Tm >90°C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 μM and 6°C but that heterodimerize with a Tm = 74°C or Kd(37) = 1.1 × 10−11 M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with Tm = 73°C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.

CODA: A combined algorithm for predicting the structurally variable regions of protein models

Abstract: CODA, an algorithm for predicting the variable regions in proteins, combines FREAD a knowledge based approach, and PETRA, which constructs the region ab initio. FREAD selects from a database of protein structure fragments with environmentally constrained substitution tables and other rule-based filters. FREAD was parameterized and tested on over 3000 loops. The average root mean square deviation ranged from 0.78 A for three residue loops to 3.5 A for eight residue loops on a nonhomologous test set. CODA clusters the predictions from the two independent programs and makes a consensus prediction that must pass a set of rule-based filters. CODA was parameterized and tested on two unrelated separate sets of structures that were nonhomologous to one another and those found in the FREAD database. The average root mean square deviation in the test set ranged from 0.76A for three residue loops to 3.09 A for eight residue loops. CODA shows a general improvement in loop prediction over PETRA and FREAD individually. The improvement is far more marked for lengths six and upward, probably as the predictive power of PETRA becomes more important. CODA was further tested on several model structures to determine its applicability to the modeling situation. A web server of CODA is available at http://www-cryst.bioc.cam.ac.uk/~charlotte/Coda/search_coda.html.

Optimization of binding electrostatics: Charge complementarity in the barnase‐barstar protein complex

Abstract: Theoretical and experimental studies have shown that the large desolvation penalty required for polar and charged groups frequently precludes their involvement in electrostatic interactions that contribute strongly to net stability in the folding or binding of proteins in aqueous solution near room temperature. We have previously developed a theoretical framework for computing optimized electrostatic interactions and illustrated use of the algorithm with simplified geometries. Given a receptor and model assumptions, the method computes the ligand-charge distribution that provides the most favorable balance of desolvation and interaction effects on binding. In this paper the method has been extended to treat complexes using actual molecular shapes. The barnase-barstar protein complex was investigated with barnase treated as a target receptor. The atomic point charges of barstar were varied to optimize the electrostatic binding free energy. Barnase and natural barstar form a tight complex (K(d) approximately 10(-14) M) with many charged and polar groups near the interface that make this a particularly relevant system for investigating the role of electrostatic effects on binding. The results show that sets of barstar charges (resulting from optimization with different constraints) can be found that give rise to relatively large predicted improvements in electrostatic binding free energy. Principles for enhancing the effect of electrostatic interactions in molecular binding in aqueous environments are discussed in light of the optima. Our findings suggest that, in general, the enhancements in electrostatic binding free energy resulting from modification of polar and charged groups can be substantial. Moreover, a recently proposed definition of electrostatic complementarity is shown to be a useful tool for examining binding interfaces. Finally, calculational results suggest that wild-type barstar is closer to being affinity optimized than is barnase for their mutual binding, consistent with the known roles of these proteins.

Crystal structure of E. coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity.

Abstract: Carbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β-class carbonic anhydrases (β-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β-CA from Escherichia coli, to a resolution of 2.0 A. In agreement with the structure of the β-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β-CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a β-CA-deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.

TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales.

Abstract: Based on the principle of dual prediction by segment hydrophobicity and nonpolar phase helicity, in concert with imposed threshold values of these two parameters, we developed the automated prediction program TM Finder that can successfully locate most transmembrane (TM) segments in proteins. The program uses the results of experiments on a series of host-guest TM segment mimic peptides of prototypic sequence KK AAAXAAAAAXAAWAAXAAAKKKK-amide (where X = each of the 20 commonly occurring amino acids) through which an HPLC-derived hydropathy scale, a hydrophobicity threshold for spontaneous membrane insertion, and a nonpolar phase helical propensity scale were determined. Using these scales, the optimized prediction algorithm of TM Finder defines TM segments by first searching for competent core segments using the combination of hydrophobicity and helicity scales, and then performs a gap-joining operation, which minimizes prediction bias caused by local hydrophilic residues and/or the choice of window size. In addition, the hydrophobicity threshold requirement enables TM Finder to distinguish reliably between membrane proteins and globular proteins, thereby adding an important dimension to the program. A full web version of the TM Finder program can be accessed at http://www.bioinformatics-canada.org/TM/.

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions.

Abstract: Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible δ-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin π-cation of compound I.

LiveBench‐1: Continuous benchmarking of protein structure prediction servers

Abstract: We present a novel, continuous approach aimed at the large-scale assessment of the performance of available fold-recognition servers. Six popular servers were investigated: PDB-Blast, FFAS, T98-lib, GenTHREADER, 3D-PSSM, and INBGU. The assessment was conducted using as prediction targets a large number of selected protein structures released from October 1999 to April 2000. A target was selected if its sequence showed no significant similarity to any of the proteins previously available in the structural database. Overall, the servers were able to produce structurally similar models for one-half of the targets, but significantly accurate sequence-structure alignments were produced for only one-third of the targets. We further classified the targets into two sets: easy and hard. We found that all servers were able to find the correct answer for the vast majority of the easy targets if a structurally similar fold was present in the server's fold libraries. However, among the hard targets—where standard methods such as PSI-BLAST fail—the most sensitive fold-recognition servers were able to produce similar models for only 40% of the cases, half of which had a significantly accurate sequence-structure alignment. Among the hard targets, the presence of updated libraries appeared to be less critical for the ranking. An “ideally combined consensus” prediction, where the results of all servers are considered, would increase the percentage of correct assignments by 50%. Each server had a number of cases with a correct assignment, where the assignments of all the other servers were wrong. This emphasizes the benefits of considering more than one server in difficult prediction tasks. The LiveBench program (http://BioInfo.PL/LiveBench) is being continued, and all interested developers are cordially invited to join.

A comparison of salts for the crystallization of macromolecules

Abstract: Thirty-one proteins and viruses that we knew from our own experience could be crystallized, or had been reported to have been crystallized by others, were investigated. In this experiment, each protein or virus was subjected to a crystallization screen of 12 different salts, each titrated to pH 7.2 beforehand, at concentrations ranging from 20% saturation to 90% saturation. Eight macromolecules failed to crystallize at all from any salt and were omitted from consideration. From the remaining 23 proteins, each salt was scored according to how many proteins and viruses it successfully crystallized. Among several results, one was particularly striking. Sodium malonate clearly was much more successful than any other salt, resulting in the crystallization of 19 of the 23 macromolecules, almost twice as effective as the next most successful salt, which was a draw between sodium acetate, sodium tartrate, sodium formate, and ammonium sulfate (11 of 22). The high success rate of sodium malonate in producing crystals was even more impressive when an overall unique success rate with individual macromolecules was considered.

Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain

Abstract: The HypF N-terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3-5 nm and 7-9 nm in width, whereas fibrils of 7-9 nm and 12-20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.

Crystal structure of Staphylococcus aureus tyrosyl-tRNA synthetase in complex with a class of potent and specific inhibitors.

Abstract: SB-219383 and its analogues are a class of potent and specific inhibitors of bacterial tyrosyl-tRNA synthetases. Crystal structures of these inhibitors have been solved in complex with the tyrosyl-tRNA synthetase from Staphylococcus aureus, the bacterium that is largely responsible for hospital-acquired infections. The full-length enzyme yielded crystals that diffracted to 2.8 A resolution, but a truncated version of the enzyme allowed the resolution to be extended to 2.2 A. These inhibitors not only occupy the known substrate binding sites in unique ways, but also reveal a butyl binding pocket. It was reported that the Bacillus stearothermophilus TyrRS T51P mutant has much increased catalytic activity. The S. aureus enzyme happens to have a proline at position 51. Therefore, our structures may contribute to the understanding of the catalytic mechanism and provide the structural basis for designing novel antimicrobial agents.

Aberrant mobility phenomena of the DNA repair protein XPA.

Abstract: The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from ∼40–45 kDa) and gel filtration chromatography (∼92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a ∼32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA ± cisplatin ± mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA ± mismatches compared to undamaged DNA ± mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from ∼5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%–50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.

Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study

Abstract: A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

Identification of intrinsic order and disorder in the DNA repair protein XPA

Abstract: The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.