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BBA - Proteins and Proteomics (v.1764, #9)

Editorial Board (pp. ii).

Microarray profiling of skeletal muscle sarcoplasmic reticulum proteins by Joseph S. Schulz; Nathan Palmer; Jon Steckelberg; Steven J. Jones; Michael G. Zeece (pp. 1429-1435).

Microarrays were developed to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum (SR) isolated from porcine Longissimus muscle. The microarrays consisted of SR preparations printed onto to glass slides and probed with monoclonal antibodies to 7 target proteins. Proteins investigated included: ryanodine receptor, (RyR), dihydropyridine receptor, (DHPR), triadin (TRI), calsequestrin (CSQ), 90 kDa junctional protein (JSR90), and fast-twitch and slow-twitch SR calcium ATPases (SERCA1 and SERCA2). Signal from a fluorescently-labeled detection antibody was measured and quantitated using a slide reader. The microarray developed was also employed to profile Longissimus muscle SR proteins from halothane genotyped animals. Significant ( P<0.05) reductions in levels of several proteins were found including: RyR, CSQ, TRI, DHPR and SERCA2 in SR samples from halothane positive animals. The results illustrate the potential of microarrays as a tool for profiling SR proteins and aiding investigations of calcium regulation.

Keywords: Protein microarray; Sarcoplasmic reticulum; Skeletal muscle; Protein profiling; Calcium regulation

Protection against glycation and similar post-translational modifications of proteins by John J. Harding; Elena Ganea (pp. 1436-1446).

Glycation and other non-enzymic post-translational modifications of proteins have been implicated in the complications of diabetes and other conditions. In recent years there has been extensive progress in the search for ways to prevent the modifications and prevent the consequences of the modifications. These areas are covered in this review together with newer ideas on possibilities of reversing the chemical modifications.

Keywords: AGE; Aminoguanidine; Aspirin; Conformation; Glycation; Post-translational modification

Role of the second-largest subunit of DNA polymerase α in the interaction between the catalytic subunit and hyperphosphorylated retinoblastoma protein in late S phase by Masaharu Takemura; Shonen Yoshida; Tetsu Akiyama; Masatoshi Kitagawa; Yoshiji Yamada (pp. 1447-1453).

DNA polymerase α (pol-α) is a heterotetrameric enzyme (p180–p68–p58–p48 in mouse) that is essential for the initiation of chain elongation during DNA replication. The catalytic (p180) and p68 subunits of pol-α are phosphorylated by Cdk–cyclin complexes, with p68 being hyperphosphorylated by cyclin-dependent kinases in G₂ phase of the cell cycle. The activity of Cdk2–cyclin A increases during late S phase and peaks in G₂ phase. We have now examined the role of p68 in the interaction between the catalytic subunit of pol-α and hyperphosphorylated retinoblastoma protein (ppRb) and in the stimulation of the polymerase activity of pol-α by ppRb. With the use of recombinant proteins, we found that nonphosphorylated p68 inhibited the stimulation of pol-α activity by ppRb, suggesting that p68 might impede the association of ppRb with p180. Phosphorylation of p68 by Cdk2–cyclin A greatly reduced its inhibitory effect. Immunofluorescence analysis also revealed that ppRb localized at sites of DNA replication specifically in late S phase. These results suggest that Cdk–cyclin A can phosphorylate pol-α which may result in a conformational change in pol-α facilitating its interaction with and activation by ppRb.

Keywords: Retinoblastoma; Rb; DNA polymerase α; Phosphorylation; Heterochromatin; Cell cycle

Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase by Lei Xu; Dongfang Zhang; Wanmeng Mu; Willem J.H. van Berkel; Zhaofeng Luo (pp. 1454-1461).

Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein–chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD_ox. Photolyase reconstituted with FAD_ox had about 34% activity of native enzyme, which increased to 83% when FAD_ox was reduced to FADH⁻. Reconstitution kinetics performed at 20 °C showed that apo-photolyase associates with FADH⁻ much faster ( k_obs ∼3000 M−¹ s−¹) than with FAD_ox ( k_obs=34 M−¹ s−¹). The dissociation constant of the photolyase–FAD_ox complex is about 2.3 μM and that of E-FADH⁻ is not higher than 20 nM (pH 7.2).

Keywords: Abbreviations; FAD; flavin adenine dinucleotide; FAD; _ox; oxidized FAD; ^FADH; neutral FAD radical; FADH; ⁻; reduced anionic FAD; MTHF; methenyltetrahydrofolate; CPD; cyclobutane pyrimidine dimer; DTT; dithiothreitolApoflavoprotein; Cofactor binding; Flavin; IMAC; Photolyase; Pterin; Reconstitution

Characterization of DitA3, the [Fe₃S₄] ferredoxin of an aromatic ring-hydroxylating dioxygenase from a diterpenoid-degrading microorganism by Manon M.-J. Couture; Vincent J.J. Martin; William W. Mohn; Lindsay D. Eltis (pp. 1462-1469).

DitA3, a small soluble ferredoxin, is a component of a ring-hydroxylating dioxygenase involved in the microbial degradation of the diterpenoid, dehydroabietic acid. The anaerobic purification of a heterologously expressed his-tagged DitA3 yielded 20 mg of apparently homogeneous recombinant protein, rcDitA3, per liter of cell culture. Each mole of purified rcDitA3 contained 2.9 equivalents of iron and 4.2 equivalents of sulfur, indicating the presence of a single [Fe₃S₄] cluster. This conclusion was corroborated by UV-Visible absorption ( ε₄₁₂=13.4 mM⁻¹cm−¹) and EPR ( g_x,y=2.00 and g_z=2.02) spectroscopies. The reduction potential of rcDitA3, determined using a highly oriented parallel graphite (HOPG) electrode, was −177.0±0.5 mV vs. the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, pH 7.0, 22 °C). This potential is similar to those of small, soluble Rieske-type ferredoxin components of aromatic-ring dihydroxylating dioxygenases. In contrast to these Rieske-type ferredoxins, DitA3 appears to exist as a dimer in solution. The dimeric ferredoxin may be more stable or may increase the catalytic efficiency of the dioxygenase by delivering the two reducing equivalents required for turnover of the oxygenase.

Keywords: [Fe; ₃; S; ₄; ] ferredoxin; Dioxygenase; Redox potential; Electrochemistry

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters by Thomas Poyot; Florian Nachon; Marie-Thérèse Froment; Mélanie Loiodice; Stacy Wieseler; Lawrence M. Schopfer; Oksana Lockridge; Patrick Masson (pp. 1470-1478).

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the₁₂₂GFYS₁₂₅ peptide segment, resulting in₁₂₂HFQT₁₂₅. This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k_cat/ K_m ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.

Keywords: Abbreviations; AChE; acetylcholinesterase; ATC; acetylthiocholine; BChE; butyrylcholinesterase; BTC; butyrylthiocholine; ChE; cholinesterase; DFP; diisopropyl phosphofluoridate; DTNB; dithio-bis-nitrobenzoic acid; OP; organophosphorus; OPase; organophosphorus hydrolase; TNB; 5-thio-2-nitrobenzoic acidAcetylcholinesterase; Organophosphate hydrolase activity; Organophosphorus inhibitors; Molecular modelling; Site-directed mutagenesis; Slow-binding inhibition

Characterization of Human γ-glutamyl hydrolase in solution demonstrates that the enzyme is a non-dissociating homodimer by Leslie E. Eisele; Karen J. Chave; Andrew C. Lehning; Thomas J. Ryan (pp. 1479-1486).

Human γ-glutamyl hydrolase (hGH) is a key enzyme in the metabolism of folic acid and in the pharmacology of many antifolate drugs. hGH catalyzes removal of the poly-γ-glutamate chains of intracellular folic acid and antifolates. hGH crystallized as a homodimer with two putative active sites. However, the quaternary structure and the number of species of the enzyme in solution have not been determined. hGH has now been characterized using analytical ultracentrifugation and dynamic light scattering. HisTag fusion proteins of wild-type hGH, rat GH, and hGH expressed as a glycosylated protein were studied. Analyses of HisTag wild-type hGH were conducted over a range of protein concentrations (1.4–200μM), ionic strengths (0–1M NaCl), and pH (4.5–8.5). A single species with a molecular mass consistent with a homodimer was observed. Glycosylated hGH and HisTag rat γ-glutamyl hydrolase also formed very stable homodimers. The lack of dissociation of the dimer, the large monomer–monomer interface, and the presence of catalytically essential Tyr-36 in the homodimer interface sequences suggest that homodimer formation is required for the hGH monomer to fold into an active conformation. The conservation of hGH monomer–monomer interface sequences in other mammalian and plant γ-glutamyl hydrolase molecules suggests that they also exist as stable homodimers.

Keywords: Abbreviations; GH; γ-glutamyl hydrolase; hGH; human GH; HisTag hGH; hGH expressed as an N-terminal 6XHis fusion protein; glycosylated hGH; hGH expressed in insect cellsFolate metabolism; Antifolate; Polyglutamate; Glutamyl hydrolase; Homodimer

Association and dissociation of the cell puncturing complex of bacteriophage T4 is controlled by both pH and temperature by Subodh Kumar Sarkar; Yoko Takeda; Shuji Kanamaru; Fumio Arisaka (pp. 1487-1492).

The tail lysozyme, gp5, of bacteriophage T4 is a trimeric protein and all the subunits are nicked between Ser351 and Ala352 during assembly through processing. When subsequently heated, the resulting (gp5*)₃ (gp5C)₃ (the asterisk “*�? denotes that the intact pre-gp5 trimer has been nicked) dissociates into three gp5* (three independent N-terminal monomeric peptides, that carry lysozyme moieties at the C-termini of gp5*), and a C-terminal trimeric β-helical structure (gp5C)₃. The interaction between gp27 and gp5* during infection is sundered by reducing pH. This dissociation would be physiologically relevant because the lysozyme moieties should be free in the periplasm (where the pH is low) and would digest the peptidoglycan layer, thereby enabling the tail tube to contact the inner membrane, and probably help to form a pore for DNA injection.

Keywords: Bacteriophage T4; Tail lysozyme; Processing; Structure; Infection; Sedimentation equilibriumAbbreviations; EDTA, ethylenediaminetetraacetic acid; gp, gene product; IPTG, isopropyl-β-; d; -thiogalactopyranoside; PCR, polymerase chain reaction; PDB, protein data bank; rpm, round per minutes; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis

Influence of amino acid properties for discriminating outer membrane proteins at better accuracy by M. Michael Gromiha; Makiko Suwa (pp. 1493-1497).

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have analyzed the influence of physico-chemical, energetic and conformational properties of amino acid residues for discriminating outer membrane proteins using different machine learning algorithms, such as, Bayes rules, Logistic functions, Neural networks, Support vector machines, Decision trees, etc. We observed that most of the properties have discriminated the OMPs with similar accuracy. The neural network method with the property, free energy change could discriminate the OMPs from other folding types of globular and membrane proteins at the 5-fold cross-validation accuracy of 94.4% in a dataset of 1088 proteins, which is better than that obtained with amino acid composition. The accuracy of discriminating globular proteins is 94.3% and that of transmembrane helical (TMH) proteins is 91.8%. Further, the neural network method is tested with globular proteins belonging to 30 major folding types and it could successfully exclude 99.4% of the considered 1612 non-redundant proteins. These accuracy levels are comparable to or better than other methods in the literature. We suggest that this method could be effectively used to discriminate OMPs and for detecting OMPs in genomic sequences.

Keywords: Abbreviations; OMP; Outer membrane protein; TMH; Transmembrane helical proteinTransmembrane β-barrel; OMP; Discrimination; Protein fold; Neural network; Amino acid property; Machine learning algorithm

Predicting 3D structures of transient protein–protein complexes by homology by Petras J. Kundrotas; Emil Alexov (pp. 1498-1511).

The paper reports a homology based approach for predicting the 3D structures of full length hetero protein complexes. We have created a database of templates that includes structures of hetero protein–protein complexes as well as domain–domain structures (http://www.ces.clemson.edu/compbio/protcom), which allowed us to expand the template pool up to 418 two-chain entries (at 40% sequence identity). Two protocols were tested–a protocol based on position specific Blast search (Protocol-I) and a protocol based on structural similarity of monomers (Protocol-II). All possible combinations of two monomers (350284 pairs) in the ProtCom database were subjected to both protocols to predict if they form complexes. The predictions were benchmarked against the ProtCom database resulting to false–true positives ratios of ∼5:1 and ∼7:1 and recovery of 19% and 86%, respectively for protocols I and II. From 350284 trials Protocol-I made only ∼500 wrong predictions resulting to 0.5% error. In addition, though it was shown that artificially created domain–domain structures can in principle be good templates for modeling full length protein complexes, more sensitive methods are needed to detect homology relations. The quality of the models was assessed using two different criteria such as interfacial residues and overall RMSD. It was found that there is no correlation between these two measures. In many cases the interface residues were predicted correctly, but the overall RMSD was over 6 Å and vice versa.

Keywords: Protein–protein complex; Homology modeling; Structural superimposition; Domain structure; Sequence similarity; Structure prediction

The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases by G.G. Hoffman; S. Sona; M. Bertin; W.R. Ellington (pp. 1512-1517).

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr→Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr→His and Tyr→Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.

Keywords: Abbreviations; CK; creatine kinase; CVMtCK; Chaetopterus variopedatus; mitochondrial creatine kinase (MtCK); DLS; dynamic light scattering; Rh; hydrodynamic radius; SarMtCK; sarcomeric MtCK; ThMtCK; Tethya aurantia; MtCK; UbiMtCK; ubiquitous MtCK; %PD; polydispersityMitochondrial creatine kinase; Quaternary structure

Binding of the lipocalin C8γ to human complement protein C8α is mediated by loops located at the entrance to the C8γ ligand binding site by Brian Chiswell; Daniel J. Slade; James M. Sodetz (pp. 1518-1524).

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the membrane attack complex (MAC). C8 is composed of a disulfide-linked C8α-γ heterodimer and a noncovalently associated C8β chain. C8α and C8β are homologous to C6, C7 and C9, whereas C8γ is the only lipocalin in the complement system. Lipocalins have a core β-barrel structure forming a calyx with a binding site for a small molecule. In C8γ, the calyx opening is surrounded by four loops that connect β-strands. Loop 1 is the largest and contains Cys40 that links to Cys164 in C8α. To determine if these loops mediate binding of C8α prior to interchain disulfide bond formation in C8α-γ, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8γ. The siderocalin-C8γ chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8α. Results indicate at least three of the four loops surrounding the entrance to the C8γ calyx are involved in binding C8α. Binding near the calyx entrance suggests C8α may restrict and possibly regulate access to the C8γ ligand binding site.

Keywords: Abbreviations; αMACPF; the membrane attack complex/perforin domain of human C8α; SC; human siderocalin; NGAL; neutrophil gelatinase-associated lipocalin; RBP; retinol binding protein; TTR; transthyretin; L1, L3, L5, L7, L15 and L157; chimeric forms of SC containing loop(s) 1, 3, 5, and/or 7 from C8γ, respectivelyComplement; C8; C8α; C8γ; Lipocalin

Erratum to “Amino acid structure and characterization of a heterodimeric disintegrin from Vipera lebetina venom�? [Biochim. Biophys. Acta 1547 (2001) 51–56] by Ammar Gasmi; Najet Srairi; Sami Guermazi; Hafedh Dekhil; Habib Karoui; Mohamed ElAyeb (pp. 1525-1525).

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BBA - Proteins and Proteomics (v.1764, #9)