bluuebunny/crossref_metadata_2025 huggingface dataset:
9,9′‐spirobifluorene‐containing polycarbonates: Transparent polymers with high refractive index and low birefringence
Authors: Seto Ryota , Sato Takashi , Kojima Takahiro , Hosokawa Katsumoto , Koyama Yasuhito , Konishi Gen‐Ichi , Takata Toshikazu
Published: July 2010

Syntheses of 2,2′‐bisalcoholic group‐substituted 9,9′‐spirobifluorene monomers 2 were performed by the reaction of 2,2′‐dihydroxy‐9,9′‐spirobifluorene 2a with haloalcohols. Polycarbonates consisting of 9,9′‐spirobifluorene skeleton in the main chain (PC 4) were synthesized by the polycondensation of 2,2′‐bisalcoholic monomers 2 and triphosgene or diphenyl carbonate. PC 4 showed good thermal stability: the 5% weight loss temperature was over 330 °C under both nitrogen and air atmospheres. The glass transition temperature was in a range of 16–269 °C estimated by differential scanning calorimetry, depending on the flexibility of the alkylene or oxyethylene chains of 2. PC 4 showed high solubility toward ordinary organic solvents such as CHCl3, benzene, and THF, making possible the preparation of the flexible thin films. Very high refractive index in a range of 1.62–1.66 at 589 nm was observed although PC 4 consists only of C, H, and O atoms, whereas very low degree of birefringence was confirmed. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3658–3667, 2010
Solution properties of thermothickening copolymers bearing hydrocarbon end‐capped oxyethylene units
Authors: Zhang Xiaofei , Jiang Xiancai , Zhang Xi , Dai Hua
Published: July 2010

Novel thermothickening copolymers composed of acrylamide and a macromer bearing hydrocarbon end‐capped oxyethylene units were synthesized. Influences of polymer concentration, salt content, shear rate, and temperature on the solution behavior were investigated. The polymer solution exhibited shear‐thickening behavior at low‐to‐moderate shear rates (<50 s−1), and the shear‐thickening behavior was dependent on polymer concentration, NaCl content, and temperature. With the increase of salinity, apparent viscosity of polymer solution increased dramatically (especially at low shear rates). At higher NaCl content (>20 wt %), polymer solutions became physical gel, and the apparent viscosity increased by several orders of magnitude. The polymer solutions exhibited excellent thermothickening behavior, even at the low concentration of 0.15 wt %. The results of rheological measurements showed that the storage and loss modulus were successfully fitted to a single Maxwell element at low temperature (<60 °C). © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1799–1808, 2010
Effect of the lithium cation on the sorption and electrical properties of amphiphilic block copolymers and their composites
Authors: da Silva M. M. S. , Rieumont J. , Nogueiras C. , Sánchez R. , de Oliveira H. P.
Published: July 2010

The use of lithium cation in composites of block copolymers [polyethylene‐b‐polyethylene oxide (PE‐b‐50%PEO and PE‐b‐80%PEO)] and their derivatives was tested as a modifier of the vapor sorption and impedance of these complexes. The block copolymer PE‐b‐80%PEO was modified by oxidation of its hydroxyl end group to both a carboxylic acid group (PE‐b‐80%PEO)CH2COOH and its sodium salt (PE‐b‐80%PEO)CH2COO− Na+ for the purpose of improving its compatibility and performance as a matrix for composites. These modified copolymers were characterized by FTIR, DSC, and mass spectrometry. The sorption of water of these copolymers and their composites with lithium nitrate was also compared, as well as the electrical properties of their composites were measured by electrical impedance spectroscopy. For the composites obtained with PE‐b‐80%PEO and lithium nitrate, it was found that lithium cation plays an important role increasing the sorption rate, which is maximized for the PE‐b‐80%PEO + (21% lithium nitrate) composite. For the copolymers (PE‐b‐80%PEO)CH2COOH and (PE‐b‐80%PEO)CH2COO− Na+ and their composites, the highest sorption rate was observed for salt in the following order: COO− Na+ > COOH > OH. The PE‐b‐80%PEO + (21% lithium nitrate) composite behaves as a solid polymeric ionic conductor fitting the Williams–Landel–Ferry equation. However, both (PE‐b‐80% PEO)CH2COOH and (PE‐b‐80%PEO)CH2COO− Na+ + (21% lithium nitrate) composites fitted the Variable Range Hopping equation, indicating a conductance trend with temperature governed by a thermally activated with energy of 0.482 and 0.524 eV and not by a relaxation process. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1809–1817, 2010
Complexation of poly(acrylic acid) and poly(ethylene oxide) investigated by enhanced Rayleigh scattering method
Authors: Li Yunbo , Li Haixia , Chen Xudong , Zhu Fangming , Yang Jin , Zhu Yubin
Published: July 2010

The method of enhanced Rayleigh scattering spectroscopy (ERS) was developed to investigate the complexation of poly(acrylic acid) (PAA) and poly(ethylene oxide) (PEO) in semidilute polymer solutions. Based on the Ornstein‐Zernike equation, the relationship between macromolecular static correlation length and ERS intensity was presented. Moreover, the ERS spectra were calculated by the moving window two‐dimensional (MW2D) correlation spectroscopy to get detailed information of the polymer complexation. The results indicated that the ERS spectroscopy characteristics of the polymer mixtures have similar trend, and the ERS intensity promptly increases as the macromolecular chains contract. The increase of ERS intensity showed that the degree of complexation between PAA and PEO increases when the pH value decreases. The complexation results from the collapse of macromolecular chains, which is induced by the PAA chains contracting and the enhanced association between PAA and PEO chains because of the hydrogen bond formation. In addition, the association resulting from the complexation of PAA and PEO in solution was demonstrated by the MW2D correlation spectroscopy. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1847–1852, 2010
‘Admission into a helping plan’: a watershed between positive and negative experiences in breast cancer
Authors: Lilliehorn Sara , Hamberg Katarina , Kero Anneli , Salander Pär
Published: July 2010

Cancer patients are in an exposed situation that raises certain psychosocial needs in contact with health care. Previous studies have mainly investigated these needs by assessments on pre‐defined categories.

Objective: The purpose of the present study is, from the patients' perspective, to identify breast cancer patients' psychosocial needs, and to synthesise them in a model reflecting the core of these needs.

Methods: Seventy‐one patients treated with radiation therapy were consecutively included and repeatedly interviewed about their experiences of health care. ‘Critical incidents’ where identified from the interviews and analysed due to the similarities–differences technique in grounded theory.

Results: Four categories of needs where detected: ‘access’, ‘information’, ‘treatment’ and ‘how approached’. These categories and their properties merged into a core category—‘admission into a helping plan’. These findings are well understood in terms of attachment theory. In times of immanent danger and stress people strive to find a ‘safe haven’ to attach to. Cancer patients' ‘safe haven’ can be described as ‘a helping plan’. It is not the result of a separate patient–caregiver relationship but is created by a pattern of individual experiences from all kind of contacts with the health‐care system as a whole.

Conclusions: The presented model of patients needs as converging into ‘admission into a helping plan’ may serve as an easily comprehendible model for caregivers, guiding them to contribute to the patient's feeling of security and trust, and thus to the patient's own ‘hope work’. Copyright © 2009 John Wiley & Sons, Ltd.
Decision‐making preferences and information needs among Greek breast cancer patients
Authors: Almyroudi Augoustina , Degner Lesley F. , Paika Vassiliki , Pavlidis Nicholaos , Hyphantis Thomas
Published: July 2010

Objectives: We aimed at assessing Greek breast cancer patients' preferences for participation in treatment decision making and their information needs.

Methods: In a cross‐sectional study, 329 breast cancer patients were administered at the Control Preferences Scale, a card‐sort measurement designed to elicit preferences for participation in decision making. Information needs were assessed with Cassileth's Information Styles Questionnaire.

Results: The majority of patients (71.1%) preferred to play a passive role in treatment decision making, with most of them wanting to delegate responsibility of the decision completely to their doctor (45.3%). A collaborative role was preferred by 24%, whereas only 4.6% chose an active role. Most women expressed a general desire for as much information as possible about their illness (62.6%), but a substantial proportion (37.4%) did not want detailed information; instead, they wished to avoid awareness of bad news. Women who desired less informational details and preferred a passive role requested less frequently a mammography (p<0.001) and/or Pap test (p<0.0005) prediagnostically.

Conclusions: This study's findings showed that the proportion of patients who wanted to play a passive role in decision making is the highest reported compared to similar studies from other countries, indicating the impact of the dominating paternalistic model of the doctor–patient relationship in the Greek medical encounter. The association of desired information details and decision‐making preferences with screening for cancer procedures prediagnostically highlights the significance of providing the patients with the appropriate information and the choices available for their treatment. Copyright © 2010 John Wiley & Sons, Ltd.
Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein
Authors: Bernhards Robert C. , Jing Xing , Vogelaar Nancy J. , Robinson Howard , Schubot Florian D.
Published: July 2010

The opportunistic pathogen P. aeruginosa utilizes a type III secretion system (T3SS) to support acute infections in predisposed individuals. In this bacterium, expression of all T3SS‐related genes is dependent on the AraC‐type transcriptional activator ExsA. Before host contact, the T3SS is inactive and ExsA is repressed by the antiactivator protein ExsD. The repression, thought to occur through direct interactions between the two proteins, is relieved upon opening of the type III secretion (T3S) channel when secretion chaperone ExsC sequesters ExsD. We have solved the crystal structure of Δ20ExsD, a protease‐resistant fragment of ExsD that lacks only the 20 amino terminal residues of the wild‐type protein at 2.6 Å. Surprisingly the structure revealed similarities between ExsD and the DNA binding domain of transcriptional repressor KorB. A model of an ExsD‐DNA complex constructed on the basis of this homology produced a realistic complex that is supported by the prevalence of conserved residues in the putative DNA binding site and the results of differential scanning fluorimetry studies. Our findings challenge the currently held model that ExsD solely acts through interactions with ExsA and raise new questions with respect to the underlying mechanism of ExsA regulation.
The osmolyte trimethylamine‐N‐oxide stabilizes the Fyn SH3 domain without altering the structure of its folding transition state
Authors: Lin Sung Lun , Zarrine‐Afsar Arash , Davidson Alan R.
Published: July 2010

Trimethylamine‐N‐oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins against denaturation. Although the impact of TMAO on the folding thermodynamics of many proteins has been well characterized, far fewer studies have investigated its effects on protein folding kinetics. In particular, no previous studies have used Φ‐value analysis to determine whether TMAO may alter the structure of the folding transition state. Here we have measured the effects on folding kinetics of 16 different amino acid substitutions distributed across the structure of the Fyn SH3 domain both in the presence and absence of TMAO. The folding and unfolding rates in TMAO, on average, improved to equivalent degrees, with a twofold increase in the protein folding rate accompanied by a twofold decrease in the unfolding rate. Importantly, TMAO caused little alteration to the Φ‐values of the mutants tested, implying that this compound minimally perturbs the folding transition state structure. Furthermore, the solvent accessibility of the transition state was not altered as reflected in an absence of a TMAO‐induced change in the denaturant β factors. Through TMAO‐induced folding studies, a β factor of 0.5 was calculated for this compound, suggesting that the protein backbone, which is the target of action of TMAO, is 50% exposed in the transition state as compared to the native state. This finding is consistent with the equivalent effects of TMAO on the folding and unfolding rates. Through thermodynamic analysis of mutants, we also discovered that the stabilizing effect of TMAO is lessened with increasing temperature.
The kinemage: A tool for scientific communication
Authors: Richardson David C. , Richardson Jane S.
Published: July 2010

A “kinemage” (kinetic image) is a scientific illustration presented as an interactive computer display. Operations on the displayed kinemage respond within a fraction of a second: the entire image can be rotated in real time, parts of the display can be turned on or off, points can be identified by selecting them, and the change between different forms can be animated. A kinemage is prepared and specified by the author(s) of a journal article, in order to better communicate ideas that depend on three‐dimensional information. The kinemages are distributed as plain text files of commented display lists and accompanying explanations. They are viewed and explored in an open‐ended way by the reader using a simple graphics program, such as the one described here (called MAGE), which presently runs on Macintosh computers. A utility (called PREKIN) helps authors prepare the kinemages. Kinemages are being implemented under the auspices of the Innovative Technology Fund.
Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin
Authors: Kaminsky S.M. , Richards F.M.
Published: July 2010

Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide. An effective 3–5‐proton difference between the oxidized and reduced protein form is observed early in proton out‐exchange of the whole protein, which is independent of temperature in the range of 5–45 °C, indicating that redox‐sensitive changes are probably not due to low‐energy structural fluctuations. Medium resolution hydrogen exchange experiments have localized the redox‐sensitive amide protons to two parts of the sequence that are distant from each other in the three‐dimensional structure: the active‐site turn and the first β‐strand. The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox‐sensitive hydrogen exchange effects are observed in the peptide experiments. A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein. Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M. & Richards, F.M., 1992, Protein Sci. 1, 22–30).
Structure of a stabilizing disulfide bridge mutant that closes the active‐site cleft of T4 lysozyme
Authors: Jacobson R.H. , Matsumura M. , Faber H.R. , Matthews B.W.
Published: July 2010

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active‐site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792–794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 °C at pH 2.

The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the α‐carbons of Cys 21 and Cys 142, on opposite sides of the active‐site cleft, to move toward each other by 2.5 Å. In association with this movement, the amino‐terminal domain of the protein undergoes a rigid‐body rotation of 5.1° relative to the carboxy‐terminal domain. This rotation occurs about an axis passing through the junction of the amino‐terminal and carboxy‐terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino‐terminal domain seen previously in crystals of wild‐type lysozyme. Even though the engineered Cys 21‐Cys 142 disulfide links together the amino‐terminal and carboxy‐terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced “hinge‐bending” mobility of the amino‐terminal domain that is suggested by the crystallographic thermal parameters of wild‐type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592–2598), the engineered cross‐link substantially enhances the stability of the protein without making the folded structure more rigid.
Automated carboxy‐terminal sequence analysis of peptides
Authors: Bailey Jerome M. , Shenoy Narmada R. , Ronk Michael , Shively John E.
Published: July 2010

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 solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS‐ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J.M. & Shively, J.E., 1990, Biochemistry 29, 3145–3156). The current study is a continuation of this work and describes the construction of an instrument for automated C‐terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N.R., Bailey, J.M., & Shively, J.E., 1992, Protein Sci. 1, 58–67), the use of sodium trimethyl‐silanolate as a novel reagent for the specific cleavage of the derivatized C‐terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C‐terminal sequencing of peptides covalently attached to carboxylic acid‐modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS‐ITC, and cleavage of the derivatized C‐terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on‐line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. 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 were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not to be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C‐terminal sequence analysis of peptides.
The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc‐dependent binding to an anionic surface
Authors: Dela Raul A. Cadena , Colman Robert W.
Published: July 2010

The histidine‐glycine‐rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin‐forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 μM and 27 μM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M was 7.3 kDa (range 6.2–8.1 kDa). Based on N‐terminal sequencing, this polypeptide (l2), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 μM. The synthetic peptide HGLGHGH representing the N‐terminal of the l2 fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459–475) included within the l2 fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 μM in the absence and presence of added Zn2+ (30 μM). The specific binding of 125I‐HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 μM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I‐HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 μM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the l2 fragment was found to inhibit 50% of the 125I‐HK binding to kaolin at a concentration of 380 μM. These results suggest that residues contained within the l2 fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.
A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin
Authors: Starovasnik Melissa A. , Klevit Rachel E. , Su Dai‐Rong , Beckingham Kathy
Published: July 2010

Calmodulin is a member of the “EF‐hand” family of Ca2+‐binding proteins. It consists of two homologous globular domains, each containing two helix‐loop‐helix Ca2+‐binding sites. To examine the contribution of individual Ca2+‐binding sites to the Ca2+‐binding properties of CaM, a series of four site‐directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca2+‐binding loops has been changed to a glutamine. One‐dimensional 1H‐NMR has been used to monitor Ca2+‐induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild‐type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca2+‐binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF‐hand Ca2+‐binding loops greatly decreases the Ca2+‐binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca2+‐binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca2+‐binding affinities of the partner C‐terminal site. In both the N‐ and C‐terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C‐terminus, the conformational consequences of mutating site III or site IV are strikingly different.
Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry
Authors: Papayannopoulos I. A. , Biemann K.
Published: July 2010

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.
ββ homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation–renaturation
Authors: Holtzer Marilyn Emerson , Kidd Suzanne Greuel , Crimmins Dan L. , Holtzer Alfred
Published: July 2010

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of αα and αβ coiled coils (α/β ≈︁ 3–4/1). In some extant studies, no ββ molecules have been found. In this study, RSTm from several different preparations was disulfide cross‐linked, both preparation and cross‐linking being done under nondenaturing conditions. The cross‐linked product was assayed for the presence of ββ molecules cross‐linked at both C36 and C190 (β=β). In such cross‐linked RSTm, 3–8% β=β is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed‐phase high‐performance liquid chromatography, and a free‐solution capillary electrophoresis experiment. This percentage becomes ∼4–10% ββ when corrected for incomplete double cross‐linking and is independent of protein concentration (0.1–10.0 mg/mL), indicating that the observed ββ species are not artifacts due to intermolecular cross‐linking. Upon denaturation and subsequent renaturation either by heating to 55 °C or by incubating at 45 °C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of ββ increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811–1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926–928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134–1138).
Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes
Authors: Atkins William M. , Villafranca Joseph J.
Published: July 2010

Single tryptophan‐containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency‐domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn‐bound wild‐type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp‐57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp‐158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406–3416). In addition, Trp‐57 and Trp‐158 both exhibit an ATP‐induced increase in the relative fraction of the long lifetime component, whereas only Trp‐57 is affected by this ligand at pH 7.5. The transition‐state analogue l‐methionine‐(R, S)‐sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp‐158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the γ‐glutamylphosphate‐ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp‐158, but has no effect on Trp‐57. Similar results were obtained when ATP was added to the MSOX complex; Trp‐57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp‐158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX‐ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp‐57 loop and the central loop (Trp‐158) during several intermediate stages of catalysis.
Mast cell tryptases: Examination of unusual characteristics by multiple sequence alignment and molecular modeling
Authors: Johnson David A. , Barton Geoffrey J.
Published: July 2010

Tryptases are trypsin‐like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M.

Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three‐dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active‐site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline‐rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin‐binding sequence, the tryptases have 10–13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn‐linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site‐directed mutagenesis.
Inclusion of solvation free energy with molecular mechanics energy: Alanyl dipeptide as a test case
Authors: Schiffer Celia A. , Caldwell James W. , Stroud Robert M. , Kollman Peter A.
Published: July 2010

A combined force field of molecular mechanics and solvation free energy is tested by carrying out energy minimization and molecular dynamics on several conformations of the alanyl dipeptide. Our results are qualitatively consistent with previous experimental and computational studies, in that the addition of solvation energy stabilizes the C5 conformation of the alanyl dipeptide relative to the C7.
An optimization approach to predicting protein structural class from amino acid composition
Authors: Zhang Chun‐Ting , Chou Kuo‐Chen
Published: July 2010

Proteins are generally classified into four structural classes: all‐α proteins, all‐β proteins, α+β proteins, and α/β proteins. In this article, a protein is expressed as a vector of 20‐dimensional space, in which its 20 components are defined by the composition of its 20 amino acids. Based on this, a new method, the so‐called maximum component coefficient method, is proposed for predicting the structural class of a protein according to its amino acid composition. In comparison with the existing methods, the new method yields a higher general accuracy of prediction. Especially for the all‐α proteins, the rate of correct prediction obtained by the new method is much higher than that by any of the existing methods. For instance, for the 19 all‐α proteins investigated previously by P.Y. Chou, the rate of correct prediction by means of his method was 84.2%, but the correct rate when predicted with the new method would be 100%! Furthermore, the new method is characterized by an explicable physical picture. This is reflected by the process in which the vector representing a protein to be predicted is decomposed into four component vectors, each of which corresponds to one of the norms of the four protein structural classes.
Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry
Authors: Aebersold Ruedi , Bures Edward J. , Goghari Mahesh H. , Namchuk Mark , Shushan Bori , Covey Tom C.
Published: July 2010

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion‐evaporation mass spectrometry. We demonstrate that the reagent 3‐[4′(ethylene‐N, N, N‐trimethylamino)phenyl]‐2‐isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion‐evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.
Renaturation of citrate synthase: Influence of denaturant and folding assistants
Authors: Zhi Wang , Landry Samuel J. , Gierasch Lila M. , Srere Paul A.
Published: July 2010

Citrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants – bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol – promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586–1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, <5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE‐assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA‐assisted refolding, this system will be useful in future studies on the mechanism of GroE‐facilitated refolding.
Extreme pKa displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes
Authors: Lederer Florence
Published: July 2010

Flavocytochrome b2 (or l‐lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related α‐hydroxy acid‐oxidizing FMN‐dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate α‐proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three‐dimensional structure of the enzyme leads to the conclusion that the high pK4 belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three‐dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the α‐hydroxy acid‐oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.
Characterization of EntF as a serine‐activating enzyme
Authors: Reichert Janice , Sakaitani Masahiro , Walsh Christopher T.
Published: July 2010

EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N‐dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to >90% homogeneity. The enzyme has been shown to complement the entF− MK1 strain in the synthesis of 2,3‐dihydroxybenzoylserine derivatives and exhibits l‐serine‐dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min−1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min−1. These results suggested the presence of an enzyme‐bound intermediate, which has been shown by gel filtration analysis to be (l‐serine)adenylate.
Convergent evolution of similar enzymatic function on different protein folds: The hexokinase, ribokinase, and galactokinase families of sugar kinases
Authors: Bork Peer , Sander Chris , Valencia Alfonso
Published: July 2010

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three‐dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three‐dimensional structure, and comprises pro‐ and eukaryotic ribokinases, bacterial fructokinases, the minor 6‐phosphofructokinase 2 from Escherichia coli, 6‐phosphotagatokinase, 1‐phosphofructokinase, and, possibly, inosine‐guanosine kinase. The third family, also of unknown three‐dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases.

Each of the three families of sugar kinases appears to have a distinct three‐dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three‐dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.
Disulfide bonding patterns and protein topologies
Authors: Benham Craig J. , Saleet Jafri M.
Published: July 2010

This paper examines the topological properties of protein disulfide bonding patterns. First, a description of these patterns in terms of partially directed graphs is developed. The topologically distinct disulfide bonding patterns available to a polypeptide chain containing n disulfide bonds are enumerated, and their symmetry and reducibility properties are examined. The theoretical probabilities are calculated that a randomly chosen pattern of n bonds will have any combination of symmetry and reducibility properties, given that all patterns have equal probability of being chosen. Next, the National Biomedical Research Foundation protein sequence and Brookhaven National Laboratories protein structure (PDB) databases are examined, and the occurrences of disulfide bonding patterns in them are determined. The frequencies of symmetric and/or reducible patterns are found to exceed theoretical predictions based on equiprobable pattern selection. Kauzmann's model, in which disulfide bonds form during random encounters as the chain assumes random coil conformations, finds that bonds are more likely to form with near neighbor cysteines than with remote cysteines. The observed frequencies of occurrence of disulfide patterns are found here to be virtually uncorrelated with the predictions of this alternative random bonding model. These results strongly suggest that disulfide bond pattern formation is not the result of random factors, but instead is a directed process.

Finally, the PDB structure database is examined to determine the extrinsic topologies of polypeptides containing disulfide bonds. A complete survey of all structures in the database found no instances in which two loops formed by disulfide bonds within the same polypeptide chain are topologically linked. Similarly, no instances are found in which two loops present on different polypeptide chains in a structure are catenated. Further, no examples of topologically knotted loops occur. In contrast, pseudolinking has been found to be a relatively frequent event. These results show a complete avoidance of nontrivial topological entanglements that is unlikely to be the result of chance events. A hypothesis is presented to account for some of these observations.
Control of nucleation in the crystallization of lysozyme
Authors: Chayen Naomi E. , Radcliffe Jonathan W. , Blow David M.
Published: July 2010

This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg‐white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze‐dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.
Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome
Authors: Chen Longyin , Mathews F. Scott , Davidson Victor L. , Tegoni Mariella , Rivetti Claudio , Rossi Gian Luigi
Published: July 2010

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c‐type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C2221 with cell dimensions a = 148.81 Å, b = 68.85 Å, and c = 187.18 Å. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper‐free apo‐amicyanin. The diffraction data were collected at 2.75 Å resolution from the former and at 2.4 Å resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD‐MADH) is an H2L2 hetero‐tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero‐octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers — i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c551i — are presented.
Mapping antibody binding sites on cytochrome c with synthetic peptides: Are results representative of the antigenic structure of proteins?
Authors: Schwab Christian , Twardek Andrea , Bosshard Hans Rudolf , Lo Terence P. , Brayer Gary D.
Published: July 2010

Crystallographic work on antigen‐antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti‐protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso‐1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti‐cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso‐1 cytochrome c and on apo‐cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apocytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c‐specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation‐dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.