bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA
Authors: Geiselmann Johannes , Yager Thomas D. , Von Hippel Peter H.
Published: February 2009

The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho‐dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n ≥ 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds ∼70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo(rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA‐dependent ATPase activity of rho. Based on these features of the rho‐RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.
Theory of chaperonin action: Inertial model for enhancement of prokaryotic rubisco assembly
Authors: Roy Harry , Kupferschmid Michael , Bell Jeffrey A.
Published: February 2009

We have performed a computational simulation of the aggregation and chaperonin‐dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P. Goloubinoff et al. (1989, Nature 342, 884–889) and P.V. Viitanen et al. (1990, Biochemistry 29, 5665–5671). The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco‐I, with itself and with aggregates of Rubisco‐I, leading up to dodecamers. Four rate constants, applying to forward or reverse steps in the aggregation process, were included. Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G. & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp. 315–322). Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one. The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al. (1989). The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin. This is followed by self‐association of the folded Rubisco monomer to yield the active dimeric Rubisco. A modification of the model that simulates temperature effects was also constructed. The most important results we obtained indicate that the chaperonin‐dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin. In addition, the simulations predict values for the association rate constant of Rubisco‐I with the chaperonin, and other variables, that are subject to experimental verification.
Analysis of peptide mixtures by capillary high performance liquid chromatography: A practical guide to small‐scale separations
Authors: Davis Michael T. , Lee Terry D.
Published: February 2009

Capillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5–50 μL/min. Detailed procedures are given for the construction of columns and UV detector flow cells. A mixture of peptides derived from the endo Lys C digest of horse heart cytochrome c was used to illustrate various aspects of capillary chromatography of peptides and compare the performance of various‐sized capillary columns and UV detector flow cell types.
Overexpression, purification, and characterization of yeast cyclophilins A and B
Authors: Zydowsky Lynne D. , Ho Susanna I. , Baker C. Hunter , Walsh Christopher T. , Mcintyre Kim
Published: February 2009

Two isoforms of yeast cyclophilins, yCyPA and yCyPB, have been subcloned, expressed in Escherichia coli, and purified to homogeneity. The full‐length (163‐amino acid) yeast CyPA was easily expressed and purified; however, only a genetically truncated, 186‐residue form of yCyPB lacking a putative 20‐amino acid signal sequence could be purified. Each yeast cyclophilin isoform is a peptidyl‐prolyl isomerase, inhibitable by the immunosuppressive drug CsA (IC50's of 40 ± 8 nM and 101 ± 14 nM at 18 nM concentrations of yCyPA and yCyPB, respectively). Polyclonal antibodies raised against recombinant yCyPA detected native yCyPA in yeast cell extracts by both immunoprecipitation and Western blot analysis. However, polyclonal antibodies raised against recombinant yCyPB detected no native yCyPB in yeast cell extracts by Western blot analysis; small amounts of yCyPB were found in the culture broth, suggesting secretion extracellularly of this isoform. Northern analysis indicated that both yCyPA mRNA and yCyPB mRNA (at a much lower level) were detectable in cell‐free extracts. Characterization of the yeast cyclophilin proteins demonstrated that their catalytic properties and sensitivity to CsA parallel those of the human cyclophilins.
Intramolecular interactions in pancreatic ribonucleases
Authors: Kolbanovskaya Elena Y.U. , Karpeisky Marat Y.A. , Sathyanarayana Bangalore K. , Wlodawer Alexander
Published: February 2009

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters in pancreatic ribonuclease A (RNase A) has been carried out. Distance calculations for all noncovalently bonded atoms revealed that the average number of nonpolar contacts between a side chain of an amino acid and its neighbors is substantially larger if it involves hydrophobic residues rather than nonhydrophobic ones. However, the difference decreased when the number of contacts per nonpolar group and/or atom were calculated. Three main nuclei and five microclusters were identified, and their quantitative parameters were calculated. These nuclei include hydrophobic residues with a substantial number of nonpolar contacts with the environment (Phe 8, Phe 120, Phe 46, Tyr 25, Tyr 97, Ile 107, Leu 35, Ile 81, Val 54, Val 108, Met 29, Met 30). Hydrophobic nuclei of RNase A differ in shape and in composition, in the number of intranuclear contacts and of associated residues, as well as in their internal mobility. All eight cysteine residues are involved in nonpolar interactions with amino acid residues of hydrophobic nuclei. Active site amino acid residues of RNase A form a noncovalent contact network comprised of themselves, as well as of many conserved residues from hydrophobic nuclei. Sequence alignment with some other members of the RNase A family of proteins shows remarkable similarity in positions and in conservation of the main nonpolar residues, comprising cores of two (out of three) hydrophobic nuclei. A correlation was shown to exist between the average density of contacts for side‐chain atoms and the number of amino acids to be found in the appropriate positions in the sequences of related mammalian ribonucleases. However, there are certain amino acid positions in the third, smaller nucleus, which are highly variable within the family. Taking into account that this nucleus is composed of residues belonging to different elements of the secondary structure, it is likely that the mutual orientation of these elements can be somehow different for these proteins.
Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state
Authors: Leonidas D. D. , Oikonomakos N. G. , Papageorgiou A. C. , Sotiroudis T. G.
Published: February 2009

R‐state monoclinic P21 crystals of phosphorylase have been shown to be catalytically active in the presence of an oligosaccharide primer and glucose‐1‐phosphate in 0.9 M ammonium sulfate, 10 mM β‐glycerophosphate, 0.5 mM EDTA, and 1 mM dithiothreitol, the medium in which the crystals are grown or equilibrated for crystallographic studies (Barford, D. & Johnson, L.N., 1989, Nature 360, 609–616; Barford, D., Hu, S.‐H., & Johnson, L.N., 1991, J. Mol. Biol. 218, 233–260). Kinetic data suggest that the activity of crystalline tetrameric phosphorylase is similar to that determined in solution for the enzyme tetramer. However, large differences were found in the maximal velocities for both oligosaccharide or glucose‐1‐phosphate substrates between the soluble dimeric and crystalline tetrameric enzyme.
Structure of a rapidly formed intermediate in ribonuclease T1 folding
Authors: Kiefhaber Thomas , Schmid Franz Xaver , Willaert Kathrien , Engelborghs Yves , Chaffotte Alain
Published: February 2009

Kinetic intermediates in protein folding are short‐lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa‐Pro peptide bonds the final rate‐limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead‐time of stopped‐flow mixing (15 ms), indicating a fast formation of the single α‐helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead‐times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the β‐sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38‐proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (τ = 6,500 s at pH 5.0 and 10 °C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native‐like properties.
Correlation functions as a tool for protein modeling and structure analysis
Authors: BÖhm Gerald , Jaenicke Rainer
Published: February 2009

Proteins present unique folding structures whose conformations are determined primarily by their amino acid sequences. At present, there is no algorithm that would correlate the sequences with the structures determined by X‐ray analysis or NMR. Comparative modeling of a new protein sequence based on the known structure of a functionally related protein promises to yield model structures that may provide relevant properties of the protein. To analyze the quality of a model structure, a set of correlation functions was derived from calculations on a subset of proteins from the structure database. Twenty‐three highly resolved protein structures with resolutions of at least 1.7 Å from various protein families were used as the primary database. The purpose of this initial work was to find highly sensitive functions (including statistical error limits for the parameters) that describe properties of “real” proteins. Each correlation described is characterized by the correlation coefficient, the parameters for linear or nonlinear regression (coefficients of the equation), standard deviation and variance, and the confidence limits describing the statistical probability for values to occur within these limits, e.g., the natural variability of the property under examination. In addition, a method was developed for creating reasonably misfolded proteins. The ability of a correlation function to discriminate between the native structure and the misfolded conformations is expressed by the reliability index, which indicates the sensitivity of a correlation function. The term correlation functions thus summarizes a variety of efforts to find a mathematical description for the properties of protein structures, for their correlation, and for their significance.
Disulfide bridges in tomato pectinesterase: Variations from pectinesterases of other species; conservation of possible active site segments
Authors: Markoviĉ Oskar , Jörnvall Hans
Published: February 2009

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys‐98 with Cys‐125, and Cys‐166 with Cys‐200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18–33% residue identities) from microorganisms. However, one half‐Cys (i.e., Cys‐166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N‐terminal part of the tomato pectinesterase, another in the C‐terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to highlight a functional site in a midchain segment.
Inactivation and covalent modification of CTP synthetase by thiourea dioxide
Authors: Robertson James G. , Sparvero Louis J. , Villafranca Joseph J.
Published: February 2009

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubations at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second‐ order rate constant for inactivation was 0.18 M−1 s−1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed‐function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.
Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K
Authors: Wong Wah Y. , Irvin Randall T. , Paranchych William , Hodges Robert S.
Published: February 2009

The C‐terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain‐specific monoclonal antibody PK99H, which blocks pilus‐mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134–140 (Asp‐Glu‐Gln‐Phe‐Ile‐Pro‐Lys) by using a single alanine replacement analysis on the 17‐residue synthetic peptide corresponding to the PAK C‐terminal sequence 128–144. Indeed, a 7‐residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17‐residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe‐Ile‐Pro‐Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain‐specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122–4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain‐specificity of PK99H for strains PAO, K122–4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain‐specificity of PK99H. Similarly, cross‐reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.
Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design
Authors: Tuchscherer G. , Servis C. , Corradin G. , Blum U. , Rivier J. , Mutter M.
Published: February 2009

The design, total chemical synthesis, and immunological properties of a four‐α‐helix bundle template‐assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58–74 of the α1 heavy chain domain of HLA‐A2 was modeled in order to increase helix stability and amphiphilicity of the 17‐mer peptide, preserving the residues for potential T‐cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four‐helix‐bundle topology of the assembled peptide blocks.

After extensive HPLC purification, stepwise solid‐phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial α‐helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four‐α‐helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA‐A2‐positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.
Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA‐binding domain as studied by amide proton exchange
Authors: Mau Ted , Baleja James D. , Wagner Gerhard
Published: February 2009

Backbone amide proton exchange rates in the DNA‐binding domain of GAL4 have been determined using 1H–15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied — the native Zn‐containing protein, the Cd‐substituted protein, and a Zn‐GAL4/DNA complex. Exchange rates in the Zn‐containing protein are significantly slower than in the Cd‐substituted protein. This shows that Cd‐substituted GAL4 is destabilized relative to the native Zn‐containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA‐recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA‐recognition module of GAL4 is reflected by the backbone amide proton exchange rates.
Electronic and vibrational spectroscopy of the cytochrome c: Cytochrome c oxidase complexes from bovine and Paracoccus denitrificans
Authors: Lynch Stephen R. , Copeland Robert A.
Published: February 2009

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein‐chromophore systems, we suggest that the energy of the Soret π* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) π‐π interactions between the heme and aromatic amino acid residues.
A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41
Authors: Fujii Gary , Horvath Suzanna , Woodward Susan , Eiserling Fred , Eisenberg David
Published: February 2009

The mechanism of protein‐mediated membrane fusion and lysis has been investigated by solution‐state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C‐terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide from bee venom, melittin. Other properties studied included the changes in visible absorbance and mean particle size, and the secondary structure of peptides as judged by CD spectroscopy. Taken together, the observations suggest that protein‐mediated membrane fusion is dependent not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form an amphiphilic structure that promotes the mixing of the lipids between membranes. A speculative molecular model is proposed for membrane fusion by α‐helical peptides, and its relationship to the forces involved in protein‐membrane interactions is discussed.
Structure of the complex between trypanosomal triosephosphate isomerase and N‐hydroxy‐4‐phosphono‐butanamide: Binding at the active site despite an “open” flexible loop conformation
Authors: Verlinde Christophe L.M.J. , Witmans Cornelis J. , Pijning Tjaard , Kalk Kor H. , Hol Wim G.J. , Callens Mia , Opperdoes Fred R.
Published: February 2009

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N‐hydroxy‐4‐phosphono‐butanamide was determined by X‐ray crystallography to a resolution of 2.84 å. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5‐å shift of the anion‐binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P‐CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.
A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I
Authors: Slupsky Carolyn M. , Shaw Gary S. , Campbell A. Patricia , Sykes Brian D.
Published: February 2009

The troponin I peptide Nα‐acetyl TnI (104–115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S., 1981, J. Biol. Chem. 256, 2798–3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726–1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974–9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C‐terminal domain of troponin C (TnC) and to calcium‐saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C‐terminal domain hydrophobic pocket and antiparallel β‐sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 + 37 μM) was found to be similar to TnIp binding to TnC (48 + 18 μM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674–681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C‐terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C‐terminal domain of TnC in structure and functional interactions.
Functional mapping of the surface of escherichia coli ribose‐binding protein: Mutations that affect chemotaxis and transport
Authors: Binnie1 R. Alan , Zhang Huide , Mowbray Sherry , Hermodson Mark A.
Published: February 2009

Ribose‐binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose‐binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.
Engineered plant phosphorylase showing extraordinarily high affinity for various α‐glucan molecules
Authors: Mori Hiroyuki , Tanizawa Katsuyuki , Fukui Toshio
Published: February 2009

α‐Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78‐residue insertion located beside the mouth of the active‐site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78‐residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C‐ and N‐terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78‐residue insertion and its flanking regions are replaced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574–5581), but when two and four unconserved residues in the N‐terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates. Finally, a chimeric phosphorylase, in which a 112‐residue sequence of the rabbit muscle enzyme involving the glycogen‐storage site was substituted for the 78‐residue insertion and its flanking regions of the type L isozyme, showed extraordinarily high affinity for a variety of substrates including not only large, branched glucans but also small, linear ones.
Sites of iodination in recombinant human brain‐derived neurotrophic factor and its effect on neurotrophic activity
Authors: Rosenfeld Robert , Philo John S. , Haniu Mitsuru , Stoney Kendall , Rohde Michael F. , Wu Gay‐May , Narhi Linda O. , Wong Caroline , Boone Tom , Hawkins Nessa N. , Miller James M. , Arakawa Tsutomu
Published: February 2009

Recombinant human brain‐derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF–Tyr‐52, Tyr‐54, Tyr‐63, and Tyr‐86–only Tyr‐63 and Tyr‐86 were iodinated under the experimental conditions used. Iodination of Tyr‐63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr‐86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr‐63 and Tyr‐86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr‐52 and Tyr‐54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr‐63 and Tyr‐86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr‐86 contributes to the biological activity.
RosettaHoles: Rapid assessment of protein core packing for structure prediction, refinement, design, and validation
Authors: Sheffler Will , Baker David
Published: February 2009

We present a novel method called RosettaHoles for visual and quantitative assessment of underpacking in the protein core. RosettaHoles generates a set of spherical cavity balls that fill the empty volume between atoms in the protein interior. For visualization, the cavity balls are aggregated into contiguous overlapping clusters and small cavities are discarded, leaving an uncluttered representation of the unfilled regions of space in a structure. For quantitative analysis, the cavity ball data are used to estimate the probability of observing a given cavity in a high‐resolution crystal structure. RosettaHoles provides excellent discrimination between real and computationally generated structures, is predictive of incorrect regions in models, identifies problematic structures in the Protein Data Bank, and promises to be a useful validation tool for newly solved experimental structures.
Central quadrant procurement of radical prostatectomy specimens
Authors: Morrison Carl , Cheney Richard , Johnson Candace S. , Smith Gary , Mohler James L.
Published: February 2009

Obtaining high quality fresh or frozen prostate tissue in gram amounts promotes the research of prostate cancer. Due to the inability to effectively recognize prostate cancer upon the cut section of a fresh prostate many pathology departments perform whole organ processing precluding the procurement of prostate tissue. Some sites randomly procure tissue, while others perform specialized procedures that can be difficult to duplicate. Neither of these types of procurement consistently results in gram quantities of tissue procured from each prostate with a high percentage of samples yielding tissue corresponding to cancer. In this study, we present a simplified model of prostate tissue procurement that has no impact on Gleason grade, tumor stage or margin status, requires minimal specialized processing, and obtains gram amounts of fresh tissue from each radical prostatectomy specimen with a high percentage yielding cancer tissue. Prostate 69: 770–773, 2009. © 2009 Wiley‐Liss, Inc.
Multistep filling of porous silicon with conductive polymer by electropolymerization
Authors: Fukami Kazuhiro , Sakka Tetsuo , Ogata Yukio H. , Yamauchi Takeshi , Tsubokawa Norio
Published: February 2009

The filling of porous silicon with polypyrrole by electropolymerization was investigated. The filling with polypyrrole proceeded preferentially along the porous silicon wall, leading to the formation of tubular structures. By repeating the porosification, the pore filling and the additional porosification, through‐tubes of polypyrrole were formed in macropores. The technique to form through‐tube was also applied to medium‐sized pores. A double layer with polypyrrole was produced by the repetition of porosification and pore filling twice. The immobilization of glucose oxidase was performed by electropolymerization in an aqueous solution containing glucose oxidase and pyrrole. Glucose oxidase was immobilized physically in the polypyrrole film. In the double layer, the sensitivity of glucose oxidase was measured by electrochemical oxidation of hydrogen peroxide, which was produced by the enzymatic reaction of glucose oxidase to gluconolactone. When glucose oxidase was immobilized in the upper layer, glucose was detected sensitively. On the other hand, when glucose oxidase was immobilized in the lower layer, the sensing current showed a slow and a low response. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Effects of initial conditions and growth temperature on the properties of nonpolar a ‐plane AlN grown by LP‐HVPE
Authors: Wu Jie‐Jun , Katagiri Yusuke , Okuura Kazuki , Li Da‐Bing , Miyake Hideto , Hiramatsu Kazumasa
Published: February 2009

AlN was grown on r ‐ and c ‐plane sapphire substrates by low‐pressure hydride vapor phase epitaxy (LP‐HVPE). Nitridation and buffer methods were used and compared. Results show that the buffer method is appropriate for the growth of a‐plane AlN. In‐plane stresses were measured and found to be different in the two in‐plane directions parallel and perpendicular to the AlN c‐axis. In‐plane stress anisotropy is reduced at high temperature leading to a smoother surface, partly owing to a decreased difference in the growth rates between two in‐plane directions. However, too high a temperature decreases the crystal quality of a‐plane AlN. Thus, there exists an optimal temperature range for the growth of a‐plane AlN, in which improved crystal and surface qualities can both be obtained. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Polarization effect on electronic band structure of InGaN/GaN multi‐quantum wells
Authors: Song Hooyoung , Kim Jin Soak , Kim Eun Kyu , Suh Moon Suhk , Seo Yong Gon , Hwang Sung‐Min
Published: February 2009

We have characterized the effect of piezoelectric field in strained InGaN/GaN multi‐quantum wells (MQWs) by using the deep level transient spectroscopy (DLTS). The photoluminescence peak of the quantum wells was observed at 2.612 eV roughly matched with DLTS results. And the carrier profile obtained by the relation of capacitance‐voltage showed carrier accumulation peaks representing the quantum wells. The thermal activation energy calculated from DLTS spectra decreased by about 0.12 eV with increasing the reverse bias field. This energy shift can be explained by the partial compensation of the internal piezoelectric field. In this report, we suggested the DLTS technique as a useful method to characterize the quantum confined Stark effect in InGaN/GaN MQWs structure. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
InGaN light emitting diodes for 415 nm–520 nm spectral range by plasma assisted MBE
Authors: Siekacz M , Szańkowska M. Ł. , Feduniewicz‐Zmuda A. , Smalc‐Koziorowska J. , Cywiński G. , Grzanka S. , Wasilewski Z. R. , Grzegory I. , Łucznik B. , Porowski S. , Skierbiszewski C.
Published: February 2009

In this work we study the growth of the Light Emitting Diodes (LEDs) by Plasma Assisted MBE (PAMBE). The active LEDs region was grown to cover the spectral range spanning from 415 nm to 520 nm. We demonstrate efficient LEDs with the highest optical power output of 1.5 mW and 20 mA for 415 nm. For longer wavelengths we observe a drop of the optical power. The reduction of the quantum efficiency for green emission can be related to the presence of strong built‐in piezoelectric fields or increased number of nonradiative recombinarion centers. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
GaN‐based embedded 2D photonic crystal LEDs: Numerical optimization and device characterization
Authors: Matioli Elison , Iza Michael , Choi Yong‐Seok , Wu Feng , Keller Stacia , Masui Hisashi , Hu Evelyn , Speck James , Weisbuch Claude
Published: February 2009

We present the numerical optimization of GaN‐based embedded 2D‐Photonic Crystal (PhC) LEDs based on a full 3D scattering matrix model pointing out the importance of a proper vertical design in embedded PhC structures. The growth of such structures following the numerical simulation trends is shown to be feasible. Experimental results of a bright embedded 2D‐PhC LED (9mW at 20mA) under electrical injection are reported. Angular measurements are performed to understand the efficiency in light extraction of the PhCs and the mechanisms limiting its full potential are presented. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
One‐ and two‐dimensional porous silicon photonic crystals: momentum dispersion relations and stopbands analysis
Authors: Gutman Nadav , Lubarski David , Sa'ar Amir
Published: February 2009

We report on a novel approach to analyze and measure the optical properties of one‐ and two ‐dimensional photonic crystals (PCs) in all spatial directions inlcluding the symmetry axis of the PCs. The analysis is based on calculating momentum dispersion relations between the ordinary and the Bloch components of the wavevector and finding momentum stop‐bands of the PC. This method provides a direct way to analyze the angular dependence of the reflection and the diffraction pattern of a monochromatic optical beam approaching the PC. Measurements have been performed on 1D PCs, e.g., 1D periodic structures of micro‐porous silicon and 2D periodic arrays of macro‐porous silicon. The periodicity of these PCs is about 2.5 to 4 μm so that their momentum stop‐bands fall in the mid‐infrared (IR) wavelengths. We analyze several geometries, including slabs and prisms, to map the momentum dispersion relations and stopbands associated with guided and radiation modes. Angular reflection and diffraction measurements using a tunable CO2 laser showed a good agreement with the calculated momentum dispersion relations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
A comparison of formulas for the design of cumulative sum control charts
Authors: Cox M. A. A.
Published: February 2009

A conventional cumulative sum control chart has a V‐mask where each arm makes an angle θ with the horizontal and has a lead distance d. These parameters are usually related to h (=dA tan(ϑ), A is a scaling factor) and k(=A tan(ϑ)). Two systems of formulas are examined for derivinghandkfor selected values of the in control and out of control average run lengths. This is the first time an exhaustive comparison has been made. Copyright © 2009 John Wiley & Sons, Ltd.
A Bayesian zero‐failure reliability demonstration test of high quality electro‐explosive devices
Authors: Fan Tsai‐Hung , Chang Chia‐Chen
Published: February 2009

Usually, for high reliability products the production cost is high and the lifetime is much longer, which may not be observable within a limited time. In this paper, an accelerated experiment is employed in which the lifetime follows an exponential distribution with the failure rate being related to the accelerated factor exponentially. The underlying parameters are also assumed to have the exponential prior distributions. A Bayesian zero‐failure reliability demonstration test is conducted to design forehand the minimum sample size and testing length subject to a certain specified reliability criterion. Probability of passing the test design as well as predictive probability for additional experiments is also derived. Sensitivity analysis of the design is investigated by a simulation study. Copyright © 2009 John Wiley & Sons, Ltd.
Direct dynamics study on the mechanism and the kinetics of the reaction of CH3NH2 with OH
Authors: Tian Wei , Wang Wenliang , Zhang Yue , Wang Weina
Published: February 2009

A theoretical study of the mechanism and the kinetics for the hydrogen abstraction reaction of methylamine by OH radical has been presented at the CCSD(T)/6‐311 ++G(2d,2p)//CCSD/6‐31G(d) level of theory. Our theoretical calculations suggest a stepwise mechanism involving the formation of a prereactant complex in the entrance channel and a preproduct complex in the exit channel, for the two hydrogen abstraction channels involving the methyl and amine groups. For clarity, the diagram of potential for the reaction is given. The calculated standard reaction enthalpies are −98.48 and −76.50 kJ mol−1 and barrier heights are 0.36 and 25.25 kJ mol−1, respectively. The rate constants are evaluated by means of the improved canonical variational transition state theory with small‐curvature tunneling correction (ICVT/SCT) in the temperature range of 299–3000 K. The calculated results show that the rate constants at experimentally measured temperatures are in good agreement with the experimental values. It is shown that the calculated rate constants exhibit a non‐Arrhenius behavior. Moreover, the variational effect is obvious in the calculated temperature range. The dominant product channel is to form CH2NH2 and H2O via hydrogen abstraction from the CH3 group of CH3NH2 by OH in the calculated temperature range. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2009
A feedback suppression algorithm for reliable satellite multicast based on spatial–temporal prediction of the satellite channel
Authors: Anastasopoulos Markos P. , Panagopoulos Athanasios D. , Cottis Panayotis G.
Published: February 2009

The major problem limiting the scalability of large‐scale multicast satellite networks is feedback implosion that arises whenever a large number of users simultaneously transmit feedback messages (FBMs) through the network, thus occupying a significant portion of satellite system resources. In satellite networks operating above 10 GHz, attenuation due to rain constitutes the dominant fading mechanism deteriorating the quality of service. In this paper, a novel scheme for providing large‐scale reliable multicast services through a star‐based geostationary satellite topology is presented incorporating accurate channel modeling of the propagation phenomena. The new protocol is based on the selection of an area representative that provides quick FBMs aiming at suppressing FBMs originating from the rest of the network users. The scheme provides a timely reaction to changes of either the channel conditions or the network topology by properly updating the selection of representatives. Through appropriate simulations, comparisons and examples it is demonstrated that the new approach suppresses FBMs very effectively. Copyright © 2009 John Wiley & Sons, Ltd.
Correcting for 3D distortion when using backscattered electron detectors in a scanning electron microscope
Authors: Proctor Jacob M.
Published: February 2009

A variable pressure scanning electron microscope (VPSEM) can produce a topographic surface relief of a physical object under examination, in addition to its two‐dimensional (2D) image. This topographic surface relief is especially helpful when dealing with porous rock because it may elucidate the pore‐space structure as well as grain shape and size. Whether the image accurately reproduces the physical object depends on the management of the hardware, acquisition, and postprocessing. Two problems become apparent during testing: (a) a topographic surface relief of a precision ball bearing is distorted and does not correspond to the physical dimensions of the actual sphere and (b) an image of a topographic surface relief of a Berea sandstone is geometrically tilted and topographically distorted even after standard corrections are applied. The procedure presented here is to ensure the veracity of the image, and includes: (a) adjusting the brightness and contrast levels originally provided by the manufacturer and (b) tuning the amplifiers of the backscatter detector plates to be equal to each other, and producing zero voltage when VPSEM is idle. This procedure is tested and verified on the said two physical samples. SCANNING 31: 59–64, 2009. © 2009 Wiley Periodicals, Inc.