bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of hollow crosslinked miktoarm polymer using miniemulsion as templates
Authors: Xiong De'an , Li Zhe , Ma Rujiang , An Yingli , Shi Linqi
Published: February 2009

Hollow crosslinked polymers (HCPs) were synthesized using arm first method via atom transfer radical polymerization. The polymerization process was performed in miniemulsion system, in which the macroinitiator, PEG‐Br, was in the water phase, whereas the vinyl‐monomer, 4‐vinylpyridine (4VP), and the crosslinker, DVB, were in the butanone phase. TEM images and light scattering characterization showed that the resultant polymer contained a hollow space, and the volume of the hollow space could be adjusted by changing the ratio of water to butanone. Also, hollow crosslinked Miktoarm polymers (HCMPs) were synthesized through this method when two different macroinitiators, PEG‐Br and PNIPAM‐Br, were used to coinitiate the polymerization of the vinyl‐monomer, 4VP and DVB. The 1H NMR spectra showed that the hollow polymers contained both PEG arms and PNIPAM arms. The hollow morphologies of the resultant Miktoarm polymers were the same as the HCPs. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1651–1660, 2009
Micelles possessing mixed cores and thermoresponsive shells fabricated from well‐defined amphiphilic ABC miktoarm star terpolymers
Authors: Zhang Yanfeng , Liu Hao , Dong Hefei , Li Changhua , Liu Shiyong
Published: February 2009

Amphiphilic ABC miktoarm star terpolymers consisting of polystyrene, poly(ε‐caprolactone), and poly(N‐isopropylacrylamide) arms, PS(‐b‐PNIPAM)‐b‐PCL, were synthesized via a combination of atom transfer radical polymerization, ring‐opening polymerization (ROP), and click chemistry. Difunctional PS bearing an alkynyl and a primary hydroxyl moiety at the chain end, PS‐alknyl‐OH, was prepared by reacting azido‐terminated PS with an excess of 3,5‐bis(propargyloxy)benzyl alcohol (BPBA) under click conditions. The subsequent ROP of ε‐caprolactone using PS‐alknyl‐OH macroinitiator afforded PS(‐alkynyl)‐b‐PCL copolymer bearing an alkynyl moiety at the diblock junction point. Target PS(‐b‐PNIPAM)‐b‐PCL amphiphilic ABC miktoarm star terpolymers were then prepared via click reaction between PS(‐alkynyl)‐b‐PCL and an excess of azido‐terminated PNIPAM (PNIPAM‐N3). The removal of excess PNIPAM‐N3 was accomplished by “clicking” onto alkynyl‐functionalized Wang resin. All the intermediate and final products were characterized by gel permeation chromatography, 1H NMR, and FTIR. In aqueous solution, the obtained amphiphilic ABC miktoarm star terpolymer self‐assembles into micelles possessing mixed PS/PCL cores and thermoresponsive shells, which were further characterized by dynamic laser light scattering and transmission electron microscopy. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1636–1650, 2009
Core‐shell particles with glycopolymer shell and polynucleoside core via RAFT: From micelles to rods
Authors: Pearson Samuel , Allen Nathan , Stenzel Martina H.
Published: February 2009

Amphiphilic block copolymers were synthesized via the reversible addition fragmentation chain transfer (RAFT) copolymerisation of 2‐methacrylamido glucopyranose (MAG) and 5′‐O‐methacryloyl uridine (MAU). Homopolymerisations of both monomers using (4‐cyanopentanoic acid)‐4‐dithiobenzoate (CPADB) proceeded with pseudo first order kinetics in a living fashion, displaying linear evolution of molecular weight with conversion and low PDIs. A bimodal molecular weight distribution was observed for PMAU at low conversions courtesy of hybrid behavior between living and conventional free radical polymerization. This effect was more pronounced when a PMAG macroRAFT agent was chain extended with MAU, however, in both cases, good control was attained once the main RAFT equilibrium was established. A stability study on PMAU found that its hydrolysis is diffusion controlled, and is accelerated at physiological pH compared with neutral conditions. Self‐assembly of four block copolymers with increasing hydrophobic (PMAU) block lengths produced micelles, which demonstrated an increased tendency to form rods as the PMAU block length increased. Interestingly, none of the block copolymers were surface‐active. An initial assessment of PMAU's ability to bind the nucleoside adenosine through base pairing was highly promising, with DSC measurements indicating that adenosine is fully miscible in the PMAU matrix. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1706–1723, 2009
Gynecological cancer survivors and community support services: referral, awareness, utilization and satisfaction
Authors: Beesley Vanessa L , Janda Monika , Eakin Elizabeth G , Auster Josephine F , Chambers Suzanne K , Aitken Joanne F , Dunn Jeff , Battistutta Diana
Published: February 2009

Objectives: To quantify gynecological cancer survivors' referral to, awareness of, utilization of and satisfaction with community support services, as well as the factors associated with service use.

Methods: In 2004, 802 gynecological cancer survivors, 3 months–5 years post‐diagnosis, completed a postal questionnaire (56% response rate). Descriptive statistics summarized outcome prevalences. Logistic regression models identified correlates of service utilization.

Results: Substantial proportions of women were aware of the main cancer support organization, Cancer Council Queensland (72%), and of information booklets (74%), helplines (66%), support groups (56%) and internet information (50%). Less than half were aware of other services. The most commonly used resources and services were information booklets (37%), the internet (23%), and helplines (20%). More broadly, 43% utilized information/internet support, 30% utilized psychosocial services and 27% utilized functional/practical services. Approximately one‐fifth (19%) used more than one support types. Having a health‐care provider referral, being diagnosed with lymphedema or living in northern Queensland were associated with higher odds of service use in all three of the support types. While most (86%) of those referred used a service, only a few women received referrals. Among users, satisfaction with services was high.

Conclusions: While gynecological cancer survivors accessed a variety of support, there is a need to ensure women are aware of services. Given the low prevalence of referrals and that referral was a key influence on service use, clinician education may be necessary to improve service referral. Organizations should also consider strategies to keep services high on clinicians' radars. Copyright © 2009 John Wiley & Sons, Ltd
Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes
Authors: Zhang Qiangmin , Peng Hao , Gao Feng , Liu Yiwei , Cheng Hao , Thompson John , Gao George F.
Published: February 2009

Gluconate 5‐dehydrogenase (Ga5DH) is an NADP(H)‐dependent enzyme that catalyzes a reversible oxidoreduction reaction between D‐gluconate and 5‐keto‐D‐gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. Despite the considerable amount of physiological and biochemical knowledge of Ga5DH, there is little physical or structural information available for this enzyme. To this end, we herein report the crystal structures of Ga5DH from pathogenic Streptococcus suis serotype 2 in both substrate‐free and liganded (NADP+/D‐gluconate/metal ion) quaternary complex forms at 2.0 Å resolution. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer that comprised four identical ∼29 kDa subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in the binding and orientation of substrate. The quaternary complex structure provides the first clear crystallographic evidence for the role of a catalytically important serine residue and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Detailed analysis of the crystal structures reveals important contributions of Ca2+ ions to active site formation and of specific residues at the C‐termini of subunits to tetramer assembly. Because Ga5DH is a potential target for therapy, our findings provide insight not only of catalytic mechanism, but also suggest a target of structure‐based drug design.
Random dissection to select for protein split sites and its application in protein fragment complementation
Authors: Chen Yong , Li Shuang , Chen Tingjian , Hua Hui , Lin Zhanglin
Published: February 2009

To identify protein split sites quickly, a selection procedure by using chloramphenicol acetyl transferase (CAT) as reporter was introduced to search for folded protein fragments from libraries generated by random digestion and reassembly of the target gene, which yielded an abundant amount of DNA fragments with controllable lengths. Experimental results of tryptophan synthase alpha subunit (TSα) and TEM‐1 β‐lactamase agreed well with what the literature has reported. The solubility of these fragments correlated roughly with the minimum inhibitory concentrations of the CAT fusions. The application of this dissection protocol to protein fragment complementation assay (PCA) was evaluated using aminoglycoside‐3′‐phosphotransferase I (APH(3′)‐I) as a model protein. Three nearly bisectional sites and a number of possible split points were identified, and guided by this result, four novel pairs of fragments were tested for complementation. Three out of four pairs partially restored the APH activity with the help of leucine zippers, and a truncated but active APH(3′)‐I (Δ1–25) was also found. Finally, the weakly active APH(3′)‐I‐(1–253)NZ/CZ (254–271) containing a short 18 residue tag was further improved by error‐prone PCR, and a best mutant was obtained showing a fourfold improvement after just one round of evolution. These results demonstrate that protein random dissection based on the CAT selection can provide an efficient search for protein breakage points and guide the design of fragments for protein complementation assay. Furthermore, more active fragment pairs can be achieved with the classical directed evolution approach.
Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs
Authors: Risso Valeria A. , Primo María E. , Ermácora Mario R.
Published: February 2009

B. licheniformis exo‐small β‐lactamase (ESBL) has a complex architecture with twelve α helices and a five‐stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL α helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.
X‐ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium pyrococcus furiosus
Authors: Day Michael W. , Hsu Barbara T. , Joshua‐Tor Leemor , Park Jae‐Bum , Zhou Zhi Hao , Adams Michael W. W. , Rees Douglas C.
Published: February 2009

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 °C, have been determined by X‐ray crystallography to a resolution of 1.8 å. Crystals of this rubredoxin grow in space group P212121 with room temperature cell dimensions a = 34.6 å, b = 35.5 å, and c = 44.4 å. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X‐PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 å and 2.06°, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 å and in bond angles of 1.95°. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen‐bonding network in the β‐sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino‐terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.
Redox properties of protein disulfide isomerase (dsba) from escherichia coli
Authors: Wunderlich Martina , Glockshuber Rudi
Published: February 2009

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 × 10−4 M was determined, corresponding to a standard redox potential (E'0) of DsbA of ‐0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 × 10−3 M to 3.9 × 10−5 M between pH 4 and 8.5. The redox state‐dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase “at work” during catalysis of oxidative protein folding.
Estimation of the maximum change in stability of globular proteins upon mutation of a hydrophobic residue to another of smaller size
Authors: Lee B.
Published: February 2009

Although the hydrophobic effect is generally considered to be one of the most important forces in stabilizing the folded structure of a globular protein molecule, there is a lack of consensus on the precise magnitude of this effect. The magnitude of the hydrophobic effect is most directly measured by observing the change in stability of a protein molecule when an internal hydrophobic residue is mutated to another of smaller size. Results of such measurements have, however, been confusing because they vary greatly and are generally considerably larger than expected from the transfer free energies of corresponding small molecules. In this article, a thermodynamic argument is presented to show (1) that the variation is mainly due to that in the flexibility of the protein molecule at the site of mutation, (2) that the maximum destabilization occurs when the protein at the site of mutation is rigid, in which case the value of the destabilization is approximately given by the work of cavity formation in water, and (3) that the transfer free energy approximately gives the minimum of the range of variations. The best numerical agreements between the small molecule and the protein systems are obtained when the data from the small molecule system are expressed as the molarity‐based standard free energies without other corrections.
Electrostatic stabilization in four‐helix bundle proteins
Authors: Robinson Clifford R. , Sligar Stephen G.
Published: February 2009

Charge substitutions generated by site‐directed mutagenesis at the termini of adjacent anti‐parallel α‐helices in a four‐helix bundle protein were used to determine a precise value for the contribution of indirect charge‐charge interactions to overall protein stability, and to simulate the electrostatic effects of α‐helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti‐parallel helices in the four‐helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four‐helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti‐parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti‐parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.
NMR analysis of staphylococcal nuclease thermal quench refolding kinetics
Authors: Kautz Roger A. , Fox Robert O.
Published: February 2009

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 °C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116‐Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis‐trans equilibrium. The rate of trans → cis isomerization in the nativelike nuclease intermediate is approximately 100‐fold faster than that observed in a Lys‐Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116‐Pro 117 peptide bond is comparable to that observed for other X‐Pro isomerizations.
Representing an ensemble of NMR‐derived protein structures by a single structure
Authors: Sutcliffe Michael J.
Published: February 2009

The usefulness of representing an ensemble of NMR‐derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of ϕΨ torsion angles and the distribution of χ1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the “average” of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the “best” rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue.
ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway
Authors: Craig Douglas B. , Wallace CARMICHAEL J. A.
Published: February 2009

Eukaryotic cytochrome c possesses an ATP‐binding site of substantial specificity and high affinity that is conserved between highly divergent species and which includes the invariant residue arginine91. Such evolutionary conservatism strongly suggests a physiological role for ATP binding that demands further investigation. We report the preparation of adducts of the protein and the affinity labels 8‐azido adenosine 5′‐triphosphate, adenosine 5′‐triphosphate‐2′, 3′‐dialdehyde, and 5′‐p‐fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine91‐containing site, yet the reaction of the latter, although specific, occurred elsewhere, suggesting caution is necessary in its use. None of the adducts displayed significant modification of global structure, stability, or physicochemical properties, leading us to believe that the 8‐N3‐ATP and oATP adducts are good stabilized models of the noncovalent interaction; yet modification led to significant, and sometimes pronounced, effects on biological activity. We therefore propose that the role of ATP binding to this site, which we have shown to occur when the phosphorylation potential of the system is high under the equivalent of physiological conditions, is to cause a decrease in electron flow through the mitochondrial electron transport chain. Differences in the degree of inhibition produced by differences in adduct chemistry suggest that this putative regulatory role is mediated primarily by electrostatic effects.
Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPβ, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors
Authors: Cho Hyeongjin , Krishnaraj Ravichandran , Walsh Christopher T. , Itoh Michyasu , Saito Haruo , Kitas Eric , Bannwarth Willi
Published: February 2009

The transmembrane PTPase HPTPβ differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354‐amino acid, 41‐kDa human PTPβ catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTPβ shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9‐11 residues from 1ck, src, and PLCγ with Km values of 2, 4, and 1 μM, some 40‐200‐fold lower than the other two PTPases. With kcat values of 30‐205 s−1, the catalytic efficiency, kcat/Km, of the HPTPβ 41‐kDa catalytic domain is very high, up to 5.7 × 107 M−1 s−1. The peptides corresponding to PLCγ (766‐776) and EGFR (1, 167‐1, 177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTPβ catalytic domain. While exchange of the alanine residue at the +2 position of the PLCγ (Km of 1 μM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35‐fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 μM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the ‐2 position of the EGFR peptide decreased the Km value 34‐fold to 3 μM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.
Site‐directed mutagenesis of Klebsiella aerogenes urease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis
Authors: Park Il‐Seon , Hausinger Robert P.
Published: February 2009

Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the γ subunit, H39 and H41 in β, and H134, H136, H219, H246, H312, H320, and H321 in the α subunit of the Klebsiella aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site‐directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The γH96A, βH39A, βH41A, αH312A, and αH321A mutant proteins possess activities and nickel contents similar to wild‐type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the αH134A, αH136A, and αH246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild‐type enzyme. These results are consistent with αH134, αH136, and αH246 functioning as nickel ligands. The αH219A protein is active and has nickel (∼1.9% and ∼80%, respectively, when compared to wild‐type protein) but exhibits a very high Km value (1, 100 + 40 mM compared to 2.3 + 0.2 mM for the wild‐type enzyme). These results are compatible with αH219 having some role in facilitating substrate binding. Finally, the αH320A protein (Km = 8.3 + 0.2 mM) only displays ∼0.003% of the wild‐type enzyme activity, despite having a normal nickel content. Unlike the wild‐type and αH219A ureases, this mutant protein was not inactivated by diethylpyrocarbonate (DEP), consistent with αH320 being the DEP‐reactive general base that facilitates catalysis.
Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis
Authors: Hu James C. , Sauer Robert T. , Newell Nicholas E. , Tidor Bruce
Published: February 2009

Combinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short α‐helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiae transcription factor GCN4 were randomized to these four residues in a λ repressor–leucine zipper fusion protein, resulting in 65, 536 possible residue combinations. Roughly three‐fourths of these combinations allowed stable coiled‐coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and non‐functional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix‐forming propensity and favorable intrasubunit and intersubunit charge‐charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.
Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry
Authors: Baca Manuel , Kent Stephen B. H. , Alewood Paul F.
Published: February 2009

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV‐1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' β‐turn mimic of predetermined three‐dimensional structure. The mimic β‐turn replaced residues Gly16, 17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry‐related type I' β‐turns in the native, unliganded enzyme. The β‐turn mimic‐containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16, 17‐containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the β‐turn at residues 15–18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid β‐turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a “molecular kit set” approach to protein design and synthesis.
Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore
Authors: Van Beeumen Jozef J. , Devreese Bart V. , Van Bun Stefaan M. , Hoff Wouter D. , Hellingwerf Klaas J. , Meyer Terrance E. , Cusanovich Michael A. , Mcree Duncan E.
Published: February 2009

The complete amino acid sequence of the 125‐residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be

MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV.

This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4Åresolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533‐6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%).

The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light‐induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein.

The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore‐binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+ 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.
Prediction of the three‐dimensional structures of the nerve growth factor and epidermal growth factor binding proteins (kallikreins) and an hypothetical structure of the high molecular weight complex of epidermal growth factor with its binding protein
Authors: Bax Ben , Ferguson Geraldine , Blaber Michael , Sternberg Michael J. E. , Walls Peter H.
Published: February 2009

We have predicted the three‐dimensional structures of the serine protease subunits (γ‐NGF, α‐NGF, and EGF‐BP) of the high molecular weight complexes of nerve growth factor (7S NGF) and epidermal growth factor (HMW‐EGF) from the mouse submandibular gland (from the X‐ray crystal structures of two related glandular kallikreins). The conformations of three of the six loops surrounding the active site are relatively well defined in the models of γ‐NGF and EGF‐BP, but three other loops are likely to have flexible conformations. Although the amino acid sequence of α‐NGF is closely related to those of γ‐NGF and EGF‐BP, it is catalytically inactive. Model‐building studies on α‐NGF suggested that mutations (in α‐NGF) just prior to the active site serine (195) and an unusual N‐terminal sequence are consistent with α‐NGF having a zymogen‐like conformation (similar to that in chymotrypsinogen).

An hypothetical model of the quaternary structure of HMW‐EGF has been constructed using this model of EGF‐BP and the NMR structure of murine EGF. The C‐terminal arm of EGF was modeled into the active site of EGF‐BP based on data indicating that the C‐terminal arginine of EGF occupies the S1 subsite of EGF‐BP. Data suggesting one of the surface loops of EGF‐BP is buried in the HMW‐EGF complex and symmetry constraints were important in deriving a schematic model. A molecular docking program was used to fit EGF to EGF‐BP.
Determining protein loop conformation using scaling‐relaxation techniques
Authors: Zheng Qiang , Rosenfeld Rakefet , Vajda Sandor , Delisi Charles
Published: February 2009

We recently developed a rapid loop closure algorithm in which bond lengths are scaled to constrain the ends of a segment to match a known distance and then gradually relaxed to their standard values, with boundary constraints maintained. Although the algorithm predicted the Zif286 zinc‐finger loop to within approximately 2 Å, it had a serious limitation that made its more general use tentative: it omitted the atomic environment of the loop. Here we report an extension of the algorithm to take into account the protein environment surrounding a given loop from the outset of the conformational search and show that it predicts structure with an efficiency and accuracy that could not be achieved without continuous environmental inclusion. The algorithm should be widely applicable to structure determination when complete experimental information is unavailable.
Hydrophobic core repacking and aromatic–aromatic interaction in the thermostable mutant of T4 lysozyme ser 117 → phe
Authors: Anderson D. Eric , Hurley James H. , Nicholson Hale , Baase Walter A. , Matthews Brian W.
Published: February 2009

The T4 lysozyme mutant Ser 117 → Phe was isolated fortuitously and found to be more thermostable than wild‐type by 1.1–1.4 kcal/mol. In the wild‐type structure, the side chain of Ser 117 is in a sterically restricted region near the protein surface and forms a short hydrogen bond with Asn 132. The crystal structure of the S117F mutant shows that the introduced Phe side chain rotates by about 150° about the Cα–Cβ bond relative to wild type and is buried in the hydrophobic core of the protein. Burial of Phe 117 is accommodated by rearrangements of the surrounding side chains of Leu 121, Leu 133, and Phe 153 and by main‐chain shifts, which result in a minimal increase in packing density. The benzyl rings of Phe 117 and Phe 153 form a near‐optimal edge–face interaction in the mutant structure. This aromatic–aromatic interaction, as well as increased hydrophobic stabilization and elimination of a close contact in the wild‐type protein, apparently compensate for the loss of a hydrogen bond and the possible cost of structural rearrangements in the mutant. The structure illustrates the ability of a protein to accommodate a surprisingly large structural change in a manner that actually increases thermal stability. The mutant has activity about 10% that of wild‐type, supportive of the prior hypothesis (Grütter, M.G. & Matthews, B.W., 1982, J. Mol. Biol. 154, 525–535) that the peptidoglycan substrate of T4 lysozyme makes extended contacts with the C‐terminal domain in the vicinity of Ser 117.
Effect of pH and denaturants on the folding and stability of murine interleukin‐6
Authors: Ward Larry D. , Zhang Jian Guo , Simpson Richard J. , Checkley Greg , Preston Barry
Published: February 2009

The conformation and stability of a recombinant mouse interleukin‐6 (mIL‐6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea‐gradient gel electrophoresis, and near‐ and far‐ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL‐6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL‐6, as judged by both urea‐gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0–10.0. Sedimentation equilibrium experiments indicated that mIL‐6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp‐36 and Trp‐160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17‐residue synthetic peptide, peptide H1, which corresponds to an N‐terminal region of mIL‐6 (residues Val‐27–Lys–43). The pH‐dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of α‐helix‐promoting solvents. Replacement of His‐33 with Ala‐33 in peptide H1 alleviated a significant portion of the pH‐dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His‐33 with the indole ring of Trp‐36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL‐6.
Defensins promote fusion and lysis of negatively charged membranes
Authors: Fujii Gary , Eisenberg David , Selsted Michael E.
Published: February 2009

Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike α‐helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of β‐sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic β‐sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein‐mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three‐dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.
Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor
Authors: Carney David F. , Hugli Tony E.
Published: February 2009

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C‐terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N‐terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site‐directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (β‐glucuronidase release). Mutants that contained the single residue substitutions Ile‐6 → Gly or Tyr‐13 → Gly were reduced in potency to 4–30% compared with wild‐type rC5a. Other single‐site glycine substitutions at positions Leu‐2, Ala‐10, Lys‐4, Lys‐5, Glu‐7, Glu‐8, and Lys‐14 showed little effect on C5a potency. The double mutant, Ile‐6 → Gly/Tyr‐13 → Gly, was reduced in potency to <0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in α‐helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N‐terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.
Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein
Authors: Chao Heman , Davies Peter L. , Sykes Brian D. , Sönnichsen Frank D.
Published: February 2009

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two‐dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase‐directed expression vector. The 67‐amino acid‐long gene product differed from the natural AFP by inclusion of an N‐terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis‐trans proline isomerization from the C‐terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine β‐strands are paired to form two triple‐stranded antiparallel sheets and one double‐stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C‐terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.
Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase
Authors: Daubner S. Colette , Lohse Daniel L. , Fitzpatrick Paul F.
Published: February 2009

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin‐dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C‐terminal two‐thirds are homologous, while the N‐terminal thirds are not. This is consistent with a model in which the C‐terminal two‐thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three‐dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N‐terminal domain mutant binds to heparin and is phosphorylated by cAMP‐dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C‐terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C‐terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.
Bacterial expression and characterization of the CREB bZip module: Circular dichroism and 2D 1H‐NMR studies
Authors: Santiago‐Rivera Zulma I. , Gorenstein David G. , Williams John S. , Andrisani Ourania M.
Published: February 2009

In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259–327, purified to near homogeneity, maintains the sequence‐specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259–327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259–327 for the CRE site was determined by gel retardation assays to be on the order of 10−7 M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259–327. The CD analyses of the native/modified CREB259–327 peptide demonstrated a 20% increase in the α‐helical content upon binding to the cAMP response‐element. Only a 5% increase in the α‐helical content of CREB259–327 is observed upon binding to the AP‐1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575–578). In addition, the two‐dimensional (2D) 1H‐NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.
Role of the C‐terminus in the activity, conformation, and stability of interleukin‐6
Authors: Ward Larry D. , Hammacher Annet , Zhang Jian‐Guo , Morton Craig J. , Simpson Richard J. , Weinstock Janet , Yasukawa Kiyoshi , Norton Raymond S.
Published: February 2009

Two murine interleukin‐6 (mIL‐6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183–187) at the C‐terminus (pMC5) and another with the last five residues of mIL‐6 substituted by the corresponding residues of human IL‐6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was <0.05% of mIL‐6, whereas pMC5H and mIL‐6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL‐6. Both pMC5 and pMC5H, like mIL‐6, failed to interact with recombinant soluble human IL‐6 receptor when assayed by surface plasmon resonance‐based biosensor analysis. These studies suggest that the C‐terminal seven amino acids of human IL‐6, alone, do not define species specificity for receptor binding.

A variety of biophysical techniques, as well as the binding of a conformational‐specific monoclonal antibody, indicated that the global fold of the mIL‐6 variants was similar to that of mIL‐6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr‐22 were influenced by the C‐terminal amino acids suggesting that the N‐ and C‐termini of mIL‐6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL‐6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C‐terminal amino acids of IL‐6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.