bluuebunny/crossref_metadata_2025 huggingface dataset:
Catalytically competent human and bovine ζ‐thrombin and chimeras generated from unfolded polypeptide chains
Authors: Lewis Sidney D. , Brezniak Diane V. , Shafer Jules A. , Fenton John W.
Published: February 2009

Human and bovine α‐thrombin cleaved at the B‐chain by chymotrypsin generates catalytically competent ζ‐thrombins, which are comprised of two noncovalently linked fragments: a 36‐ (human) or 49‐ (bovine) residue A‐chain linked by a disulfide to B‐chain residues B1–148 (ζ1‐thrombin) and B‐chain residues B149–259 (ζ2‐thrombin). Human and bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins were prepared by reaction of the active‐site histidine (H‐B43) and serine (S‐B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins in 4.5 M guanidine‐HCl and refolding upon 30‐fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine‐HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine ζ1‐thrombin:human ζ2‐thrombin and human ζ1‐thrombin:bovine ζ2‐thrombin. Human ζ1‐thrombin and ζ2‐thrombin were isolated, and, upon recom‐bining, the isolated fragments refolded to generate catalytically competent ζ‐thrombin with an active‐site content, specific activity toward Chromozym‐TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native α‐thrombin. Both α‐ and ζ‐thrombin refolded via first‐order processes (k = 0.011–0.014 s−1), which in the case of ζ‐thrombin were independent of whether ζ1‐thrombin and/or ζ2‐thrombin was incubated in refolding buffer prior to mixing. This observation, together with others, is consistent with the view that generation of catalytically competent enzyme proceeds via a kinetic pathway wherein ζ1‐thrombin and ζ2‐thrombin independently partially refold and form a noncovalent complex that undergoes a rate‐determining rearrangement to active thrombin.
Crystal structure of a complex of HIV‐1 protease with a dihydroxyethylene‐containing inhibitor: Comparisons with molecular modeling
Authors: Thanki Narmada , Rao J.K. Mohana , Foundling Stephen I. , Wlodawer Alexander , Howe W. Jeffrey , Moon Joseph B. , Hui John O. , Tomasselli Alfredo G. , Heinrikson Robert L. , Thaisrivongs Suvit
Published: February 2009

The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV‐1) protease with a peptide‐mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa‐His‐Hch[CH(OH)CH(OH)]Vam‐Ile‐Amp (U‐75875), and its Ki for inhibition of the HIV‐1 protease is <1.0 nM (Noa = 1‐naphthoxyacetyl, Hch = a hydroxy‐modified form of cyclohexylalanine, Vam = a hydroxy‐modified form of valine, Amp = 2‐pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 Å resolution by using restrained least‐squares procedures. Root mean square deviations from ideality are 0.02 Å and 2.4°, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X‐ray structure with a model‐built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side‐chain atoms in the P2‐P2′ region, where the interaction with the protein is strongest. However, the X‐ray structure and the model differ considerably in the location of the P3 and P3′ end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1‐P1′ geometry very close to that seen in the X‐ray structure.
Crystallization of proton channel peptides
Authors: Lovejoy Brett , Åkerfeldt Karin S. , Degrado William F. , Eisenberg David
Published: February 2009

Crystals have been grown of two similar peptides that form ion‐conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2‐trifluoroethanol. Crystals of one of the peptides have been characterized by X‐ray diffraction, and X‐ray data have been measured to 2.3 Å resolution. Earlier it was proposed that the ion‐conducting channels formed by these peptides consist of four peptides associated as a parallel α‐helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.
Control of phosphorylase b conformation by a modified cofactor: Crystallographic studies on R‐state glycogen phosphorylase reconstituted with pyridoxal 5′‐diphosphate
Authors: Leonidas D. D. , Oikonomakos N.G. , Papageorgiou A. C. , Acharya K. R. , Barford D. , Johnson L. N.
Published: February 2009

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5′‐phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5′‐diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716–6722) have shown that PLPP promotes R‐state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5′‐diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R‐state crystal form and the structure refined from X‐ray data to 2.8 Å resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R‐state GPb‐AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R‐state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T‐state structure shows that were the PLPP to be incorporated into the T‐state structure there would be a close contact with the 280s loop (residues 282–285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose‐1‐phosphate and sulfate (in the R state) and for heptulose‐2‐phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.
Molecular dynamics studies of a DNA‐binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor
Authors: Guenot Jeanmarie , Kollman Peter A.
Published: February 2009

Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein‐protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance‐dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance‐dependent and constant dielectric models becomes smaller, although the linear distance‐dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance‐dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance‐dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance‐dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance‐dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable.
Accelerated high‐sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge
Authors: Totty Nicholas F. , Hsuan J. Justin , Waterfield Michael D.
Published: February 2009

Greatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on‐line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.
Unfolding domains of recombinant fusion αα‐tropomyosin
Authors: Ishii Yoshiharu , Hitchcock‐Degregori Sarah , Mabuchi Katsuhide , Lehrer Sherwin S.
Published: February 2009

The thermal unfolding of the coiled‐coil α‐helix of recombinant αα‐tropomyosin from rat striated muscle containing an additional 80‐residue peptide of influenza virus NS1 protein at the N‐terminus (fusion‐tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion‐tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 °C involving the middle of the molecule; (2) a major transition at 46 °C involving no more than 36% of the helix from the C‐terminus; (3) a major transition at 56 °C involving about 46% of the helix from the N‐terminus; and (4) a transition from the nonhelical fusion domain at about 70 °C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 °C or 70 °C transition. The very stable fusion unfolding domain of fusion‐tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a crosslink to stabilize the adjacent N‐terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C‐terminal domain at 46 °C from the stabilized N‐terminal unfolding domain at 56 °C. Thus, strong localized interchain interactions in coiled‐coil molecules can increase the stability of neighboring domains.
A new subfamily of bacterial ABC‐type transport systems catalyzing export of drugs and carbohydrates
Authors: Reizer Jonathan , Reizer Aiala , Saier Milton H.
Published: February 2009

Sequence comparison studies revealed that the drug resistance transporter of Streptomyces peucetius (DrrAB) and two nodulation gene products (NodIJ) of Rhizobium leguminosarum are homologous to proteins encoded by three sets of genes that comprise capsular polysaccharide export systems in gram‐negative bacteria: KpsTM of Escherichia coli, BexABC of Haemophilus influenzae, and CtrDCB of Neisseria meningitidis. These five systems comprise a new subfamily within the family of ATP binding cassette (ABC)‐type transporters. We have termed this subfamily the ABC‐2 subfamily. For three of the systems comprising this subfamily (Drr, Nod, and Kps) only one integral membrane constituent has been identified, whereas for the other two systems (Bex and Ctr) two dissimilar integral membrane constituents have been found. This observation suggests that the transmembrane channels of ABC‐2‐type transporters can be formed of homo‐ or heterooligomers as is true of several other classes of transport systems.
NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase
Authors: Lee Jonathan P. , Cho Hyeongjin , Bannwarth Willi , Kitas Eric A. , Walsh Christopher T.
Published: February 2009

An autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142–1,153 of the insulin receptor was prepared and incubated with the 40‐kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H‐NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri‐, di‐, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.
Structure of the oxidized long‐chain flavodoxin from anabaena 7120 at 2 å resolution
Authors: Rao S. T. , Shaffie Fathieh , Yu Carlos , Satyshur Kenneth A. , Stockman Brian J. , Markley John L. , Sundaralingam M.
Published: February 2009

The structure of the long‐chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 å resolution by the molecular replacement method using the atomic coordinates of the long‐chain flavodoxin from Anacystis nidulans. The structure of a third long‐chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P21 with a = 48.0, b = 32.0, c = 51.6 å, and β = 92°, and one molecule in the asymmetric unit. The 2 å intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2σ and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R‐value of 0.185 for a model containing atoms for protein residues 2–169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5′‐phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long‐chain structures are similar except for the O4′ of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9–15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 å and 3.5 å, respectively, whereas in the third flavodoxin the distance is 3.1 å, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent. In all three long‐chain flavodoxins, a water molecule bridges the ends of the inserted loop in the β5 strand and minimally perturbs its hydrogen bonding with β4. Many of the water molecules in these proteins interact with the flavin binding loops. The conserved β‐core of the three long‐chain and two short‐chain flavodoxins superpose with root mean square deviations ranging from 0.48 å to 0.97 å.
Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study
Authors: Stebbins Jeffrey W. , Robertson Diane E. , Roberts Mary F. , Stevens Raymond C. , Lipscomb William N. , Kantrowitz Evan R.
Published: February 2009

The replacement of Arg‐54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000‐fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., & Kantrowitz, E.R., 1989, Biochemistry 28, 2592–2600). In the X‐ray structure of the wild‐type enzyme, Arg‐54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E. & Lipscomb, W.N., 1988, Proc. Natl. Acad. Sci. USA 85, 4205–4208). The Arg‐54 → Ala enzyme was crystallized in the presence of the transition state analog N‐phosphonoacetyl‐l‐aspartate (PALA), data were collected to a resolution limit of 2.8 å, and the structure was solved by molecular replacement. The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules. 31P‐NMR studies indicate that the binding of CP to the wild‐type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme. The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pKa ∼ 7.7 upon CP binding. Furthermore, the linewidth of the 31P signal of CP bound to the Arg‐54 → Ala enzyme is significantly narrower than that of CP bound to the wild‐type catalytic subunit at any pH, although the change in chemical shift for the CP bound to the mutant enzyme is unaltered. 31P‐NMR studies of PALA complexed to the wild‐type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg‐54 → Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8. Thus, the side chain of Arg‐54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state. These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.
X‐ray structure of glu 53 human lysozyme
Authors: Harata Kazuaki , Muraki Michiro , Hayashi Yasuhiro , Jigami Yoshifumi
Published: February 2009

The three‐dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 å resolution by X‐ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild‐type HL. The root mean square difference for the superposition of equivalent Cα atoms is 0.141 å. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild‐type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 å between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild‐type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.
Internal water molecules and H‐bonding in biological macromolecules: A review of structural features with functional implications
Authors: Meyer Edgar
Published: February 2009

Conserved structural patterns of internal water molecules and/or H‐bond chains were observed and are here correlated in this review, which then describes two functional properties: equilibration of hydrostatic pressure and proton transport. Available evidence in support of these hypotheses is presented, together with suggested experiments to test them. High‐resolution crystal structures of a variety of proteins were studied with interactive computer graphics. Conserved H‐bonding linkages may be used as a paradigm for a rationalization of proton transport in membranes. The concept of the “proton wire,” which links buried active‐site amino acids with the surface of the protein raises the more general question of the functional role of the various molecular components
Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: Increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines
Authors: Sekharudu Chandra , Ramakrishnan Boopathy , Huang Baohua , Jiang Ru‐Tai , Dupureur Cynthia M. , Tsai Ming‐Daw , Sundaralingam Muttaiya
Published: February 2009

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn‐2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp‐99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr‐52 and ‐73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X‐ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 å resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3121, with cell parameters α = b = 46.3 å and c = 102.95 å. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R‐factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the α‐carbon atoms between the double mutant and wild type was 0.56 å. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro‐68. However, the hydrogen bonds of the catalytic triad, His‐48, Asp‐99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe‐52 moves toward His‐48 and Asp‐99 of the catalytic diad, and Phe‐73 moves toward Met‐8, both by about 0.5 å. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.
Effects of calcium on recombinant bovine chromogranin A
Authors: Angeletti Ruth Hogue , Ali Ghazala , Shen Ning , Gee Patricia , Nieves Edward
Published: February 2009

Bovine chromogranin A, the acidic calcium‐binding protein characteristic of endocrine secretory vesicles, has been expressed inEscherichia coliusing the pET3a vector system under T7 polymerase control. The expressed protein is located in the bacterial cytosol and can be purified from bacterial proteins by a heat treatment step, followed by gel filtration, anion‐exchange, and reversed‐phase chromatography. The purified recombinant chromogranin A has an apparentMTof ca. 72,000 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, in spite of its 432‐amino acid polypeptide chain, consistent with observations on natural chromogranin A. The primary structure has been confirmed by mass spectral analysis of tryptic peptides, by Edman degradation of the intact protein, and by immunoreactivity with sequence‐specific antibodies. Analysis by circular dichroism spectroscopy shows pH‐ and concentration‐dependent spectra. The spectra are Ca2+‐dependent from 5 to 40 μM.
Automated carboxy‐terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate
Authors: Bailey Jerome M. , Nikfarjam Firoozeh , Shenoy Narmada R. , Shively John E.
Published: February 2009

Proteins and peptides can be sequenced from the carboxy‐terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS‐ITC) to derivatize the C‐terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C‐terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68–80). A major limitation of this approach was the need to activate the C‐terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP‐ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673–678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C‐terminal thiohydantoins by DPP‐ITC resulting in complete conversion of the C‐terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP‐ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C‐terminal carboxylic acid to a salt prior to the reaction with DPP‐ITC and pyridine. By introducing the DPP‐ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS‐ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C‐terminal sequencing of peptides covalently attached to carboxylic acid‐modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C‐terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C‐terminal sequence analysis of peptides and proteins. This work represents the first time an automated, sequential, chemical method for C‐terminal sequence analysis has been applied to subnanomolar amounts of protein samples.
Escherichia coli K12 arabinose‐binding protein mutants with altered transport properties
Authors: Kehres D.G. , Hogg R.W.
Published: February 2009

The arabinose‐binding protein (ABP) of Escherichia coli binds l‐arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose‐binding cleft were subjected to site‐directed mutagenesis. Thirty‐eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild‐type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and Ken (concentration of arabinose yielding half‐maximal entry) were determined. Twenty‐four mutants had normal Ven values, 3 mutants had Ven and Ken values twice wild type, and 11 mutants had Ven and Ken values 20–50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild‐type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose‐binding cleft, all of which are charged in the wild‐type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.
A kinetic model for binding protein‐mediated arabinose transport
Authors: Kehres D.G.
Published: February 2009

A kinetic model is presented based on the simplest plausible mechanism for bacterial binding protein‐dependent transport. The transport phenotypes of the 18 variant arabinose‐binding proteins analyzed by Kehres and Hogg (1992, Protein Sci. 1, 1652–1660) (wild type and 17 mutants) are interpreted to mean that in wild‐type arabinose uptake the forward transport rate (kfor) greatly exceeds the dissociation rate (kund) of a binding protein docked with the AraG:AraH membrane complex, and that kfor dominance is preserved in all of the binding protein surface mutants. The assumptions and predictions of the model are consistent with existing data from other periplasmic transport systems.
Stability and reconstitution of pyruvate oxidase from lactobacillus plantarum: Dissection of the stabilizing effects of coenzyme binding and subunit interaction
Authors: Risse Bernhard , Stempfer Günter , Rudolph Rainer , Möllering Hans , Jaenicke Rainer
Published: February 2009

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated.

As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size‐exclusion high‐performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 μg/mL, whereas the apoenzyme shows stepwise tetramer‐dimer‐monomer dissociation, with the monomer as the major component, at a protein concentration of <20 μg/mL.

In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect.

Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer‐tetramer equilibrium transition with an association constant of Ka = 2 × 107 M−1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 μM FAD. This association step obeys second‐order kinetics with an association rate constant k = 7.4 × 103 M−1 s−1 at 20 °C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.
In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV
Authors: Potter Steven M. , Henzel William J. , Aswad Dana W.
Published: February 2009

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein l‐isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl‐3H from S‐adenosyl‐l‐[methyl‐3H]methionine to the isoaspartyl (α‐carboxyl) side chain. More than 1.2 mol of methyl‐acceptor sites per mol of calmodulin accumulated during a 2‐week incubation without calcium at pH 7.4, 37°C. Analysis of proteolytic peptides of aged calmodulin revealed that >95% of the methylation capacity is restricted to residues in the four calcium‐binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn‐97‐Gly‐98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp‐131‐Gly‐132 and Asp‐133‐Gly‐134 in domain IV, and Asn‐60‐Gly‐61 in domain II. Significant isoaspartate formation was also localized to Asp‐20, Asp‐22, and/or Asp‐24 in domain I, to Asp‐56 and/or Asp‐58 in domain II, and to Asp‐93 and/or Asp‐95 in domain III. All of these residues are calcium ligands in the highly conserved EF‐hand calcium‐binding motif. Thus, other EF‐hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C‐flanking residue and by local flexibility.
Disulfide structures of highly bridged peptides: A new strategy for analysis
Authors: Gray William R.
Published: February 2009

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water‐soluble tris‐(2‐carboxyethyl)‐phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol–disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol–disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra‐ and interchain disulfides; ‐Cys·Cys‐ pair), endothelin and apamin (two disulfides; ‐Cys·x·Cys‐ pair), conotoxin GI and isomers (two disulfides; ‐Cys·Cys‐ pair), and bacterial enterotoxin (three bridges within 13 residues; two ‐Cys·Cys‐ pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.
Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates
Authors: Sakane Isao , Hongo Kunihiro , Mizobata Tomohiro , Kawata Yasushi
Published: February 2009

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single‐molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force‐extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm‐like chain model, the contour lengths calculated from the peaks in the profile suggested that linked‐GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.
Effects of the sesquiterpene lactone parthenolide on prostate tumor‐initiating cells: An integrated molecular profiling approach
Authors: Kawasaki Brian T. , Hurt Elaine M. , Kalathur Madhuri , Duhagon Maria Ana , Milner John A. , Kim Young S. , Farrar William L.
Published: February 2009

Recent evidence suggests tumor‐initating cells (TICs), also called cancer stem cells, are responsible for tumor initiation and progression; therefore, they represent an important cell population for development of future anti‐cancer therapies. In this study, we show that the sesquiterpene lactone parthenolide (PTL) is cytotoxic to prostate TICs isolated from prostate cancer cell lines: DU145, PC3, VCAP, and LAPC4, as well as primary prostate TICs. Furthermore, PTL inhibited TIC‐driven tumor formation in mouse xenografts. Using an integrated molecular profiling approach encompassing proteomics, profiles of activated transcription factors and genomics we ascertained the effects of PTL on prostate cancer cells. In addition to the previously described effects of PTL, we determined that the non‐receptor tyrosine kinase src, and many src signaling components, including: Csk, FAK, β1‐arrestin, FGFR2, PKC, MEK/MAPK, CaMK, ELK‐1, and ELK‐1‐dependent genes are novel targets of PTL action. Furthermore, PTL altered the binding of transcription factors important in prostate cancer including: C/EBP‐α, fos related antigen‐1 (FRA‐1), HOXA‐4, c‐MYB, SNAIL, SP1, serum response factor (SRF), STAT3, X‐box binding protein‐1 (XBP1), and p53. In summary, we show PTL is cytotoxic to prostate TICs and describe the molecular events of PTL‐mediated cytotoxicity. Therefore, PTL represents a promising therapeutic for prostate cancer treatment. Prostate 69: 827–837, 2009. © 2009 Wiley‐Liss, Inc.
Gene expression changes are age‐dependent and lobe‐specific in the brown Norway rat model of prostatic hyperplasia
Authors: Bethel Carlise R. , Chaudhary Jaideep , Anway Matthew D. , Brown Terry R.
Published: February 2009

BACKGROUND

Benign prostatic hyperplasia (BPH) is an age‐related enlargement of the prostate, characterized by increased proliferation of stromal and epithelial cells. Despite its prevalence, the etiology of BPH is unknown.

METHODS

The Brown Norway rat is a model for age‐dependent, lobe‐specific hyperplasia of the prostate. Histological analyses of the dorsal and lateral lobes from aged rats reveal focal areas characterized by increased numbers of luminal epithelial cells, whereas the ventral lobe is unaffected. This study examined differential gene expression by lobe and age in the Brown Norway rat prostate. The objective was to identify genes with different levels of expression in the prostate lobes from 4‐month (young) and 24‐month (old) animals, and to subsequently link changes in gene expression to mechanisms of prostate aging.

RESULTS

The number of age‐dependent differentially expressed genes was greatest in the dorsal compared to the ventral and lateral lobes. Minimal redundancy was observed among the differentially expressed genes in the three lobes. Age‐related changes in the expression levels of 14 candidate genes in the dorsal, lateral and ventral lobes were confirmed by quantitative RT‐PCR. Genes that exhibited age‐related differences in their expression were associated with proliferation, oxidative stress, and prostate cancer progression, including topoisomerase II alpha (Topo2a), aurora kinase B (Aurkb), stathmin 1 (Stmn1), and glutathione S‐transferase pi. Immunohistochemistry for Topo2a, Aurkb, and Stmn1 confirmed age‐related changes in protein localization in the lateral lobe of young and aged prostates.

CONCLUSION

These findings provide clues to the molecular events associated with aging in the prostate. Prostate 69: 838–850, 2009. © 2009 Wiley‐Liss, Inc.
Lab‐on‐substrate technology platform
Authors: Braun Tanja , Böttcher Lars , Bauer Jörg , Jung Erik , Becker Karl‐Friedrich , Ostmann Andreas , Aschenbrenner Rolf , Reichl Herbert
Published: February 2009

A technology platform based on standard PCB technology has been developed for cost‐effective fabrication of biological and medical test devices. Aluminum, polyimide and Pyralux have been selected as materials with focus on bio‐ and process compatibility. A process flow consisting of lamination, Al structuring by wet etching, microwell and via formation by laser drilling and via metallization was developed. Surface treatments have been evaluated for hydrophobic and hydrophilic modification of the materials. The technology was demonstrated with a dielectrophoresis enhanced microwell device for single cell handling and detection of cell‐to‐cell interaction as needed for the improvement of tumor therapy. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Oxide removal from GaN(0001) surfaces
Authors: Schulz Ch. , Schmidt Th. , Flege J. I. , Berner N. , Tessarek Ch. , Hommel D. , Falta J.
Published: February 2009

X‐ray photoelectron spectroscopy has been used to study two different two‐step cleaning methods of MOVPE grown GaN(0001). Carbon and oxygen were identified as the major contaminants, even though the samples were stored in a glove box after growth and transferred under nitrogen atmosphere. By thermal annealing of the samples a significant oxygen decrease and a complete removal of the carbon was achieved. To remove the residual oxygen, subsequently Ga deposition/re‐desorption cycles and N‐plasma treatment were performed (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Thermal characterization of light‐emitting diodes in the frequency domain
Authors: Vitta Pranciškus , Žukauskas Artūras
Published: February 2009

We report on a method for measurement of thermal relaxation time constants within light‐emitting diodes (LEDs) using harmonic modulation of the driving current. The method is based on the phase shift of the forward voltage waveform in respect to that of the modulated current. The phase shift is due to the sensitivity of the forward voltage to junction temperature, which responds to the modulation of the heat generation depending on the thermal relaxation rate. The frequency dependence of the phase shift was shown to exhibit characteristic dips at angular frequencies equal to inverse thermal time constants. Such an approach for thermal characterization was demonstrated for common GaP, AlGaAs, AlInGaP, and InGaN LEDs. In particular, low‐power p‐n and double‐heterostructure LEDs as well as high‐power truncated‐inverted‐pyramid and flip‐chip LEDs were investigated. The measured thermal time constants (∼ 0.1–100 ms) were tentatively assigned to heat flows within the multilayer structure of the LEDs and collated with the numerical estimates based on thermal resistance and heat capacitance of the LED components. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Influence of compositional changes of source materials on AlN synthesis using Li‐Al‐N solvent
Authors: Nagano T. , Kangawa Y. , Kakimoto K.
Published: February 2009

We carried out AlN synthesis using Li‐Al‐N solvent. In the present work, Al and Li3N were used as source materials. In this paper, we discuss the variation in phase stability of the products with change in Li‐Al‐N composition, i.e., Al/Li3N molar ratio. The results suggest that LiAlO2 extra phase was formed in the products under an Al‐rich synthesis condition. The oxide formation seems to be caused by oxidation of the nitrogen source. That is, some of the Li3N reacted with H2O in the ambient and to be LiOH, though preparation of the source materials was performed in a glove‐box. On the other hand, scanning electron microscopy (SEM) images indicate the possibility of reducing nucleation sites and increasing grain size by optimizing the Al/Li3N molar ratio. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)