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BBA - Proteins and Proteomics (v.1824, #2)

Editorial Board (pp. i).

QM/MM study of the mechanism of enzymatic limonene 1,2-epoxide hydrolysis by Q.Q. Hou; X. Sheng; J.H. Wang; Y.J. Liu; C.B. Liu (pp. 263-268).

Limonene 1,2-epoxide hydrolase (LEH) is completely different from those of classic epoxide hydrolases (EHs) which catalyze the hydrolysis of epoxides to vicinal diols. A novel concerted general acid catalysis step involving the Asp101–Arg99–Asp132 triad is proposed to play an important role in the mechanism. Combined quantum-mechanical/molecular-mechanical (QM/MM) calculations gave activation barriers of 16.9 and 25.1kcal/mol at the B3LYP/6-31G(d,p)//CHARMM level for nucleophilic attack on the more and less substituted epoxide carbons, respectively. Furthermore, the important roles of residues Arg99, Tyr53 and Asn55 on mutated LEH were evaluated by QM/MM-scanned energy mapping. These results may provide an explanation for site-directed mutagenesis.The final solvated model of LEH after QM/MM optimization is shown in the left panel, and the optimized structure of the reactive pocket is shown in the right panel. As can be seen from the right panel, there is a large hydrogen bonding network in the active pocket, and the substrate presents favorable direction for nucleophilic attack by the catalytic water molecule.Display Omitted►We carried out QM/MM calculations to elucidate the reaction mechanism of LEH. ►The nucleophilic attack on the more substituted carbon is favorable. ►Arg99, Tyr53 and Asn55 play an important role in the catalytic reaction.

Keywords: QM/MM; Hydrolases; Epoxide ring-opening; Reaction mechanism; Mutation

An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification by Anna C. Nelson Dittrich; Timothy P. Devarenne (pp. 269-273).

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the “gatekeeper” residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3^M385G can also specifically utilize N⁶-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.► An analog sensitive form of the plant cell death suppressor kinase Adi3 is developed. ► Use of bulky ATP analogs to identify kinase substrates. ► Adi3 Met385 mutation allows for use of bulky ATP analogs for phosphotransfer.

Keywords: Abbreviations; Adi3; AvrPto-dependent Pto-interacting protein 3; CDK2; cyclin-dependent kinase 2; Gal83; galactose-specific gene 83; MBP; maltose binding protein; NDPK; nucleoside diphosphate kinase; PCD; programmed cell death; PKA; protein kinase A (or cAMP-dependent protein kinase); SnRK1; Sucrose non-Fermenting-1-Related Protein Kinase 1; v-Src; viral sarcomaAdi3; Analog-sensitive kinase; Programmed cell death

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone by Maria Andreasen; Søren B. Nielsen; Tina Mittag; Morten Bjerring; Jakob T. Nielsen; Shuai Zhang; Erik H. Nielsen; Martin Jeppesen; Gunna Christiansen; Flemming Besenbacher; Mingdong Dong; Niels Chr. Nielsen; Troels Skrydstrup; Daniel E. Otzen (pp. 274-285).

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6–10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe–Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the −NH− groups has been replaced by −CH₂−) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.Display Omitted► We examine backbone contribution to fibril stability using modified peptides. ► The backbone is extremely sensitive to substitutions. ► One analogue could elongate fibril seeds, none could fibrillate on their own. ► The resulting fibrils display differences in secondary structure and morphology. ► Side-chain mutations had little effect on fibrillation kinetics and stability.

Keywords: Abbreviations; GAVL; decapeptide SNNFGAVLSS; GGIL; decapeptide SNNFGGILSS; NF6; hexapeptide NFGAIL; SN10; decapeptide SNNFGAILSS; ThT; thioflavin TAmylin; Peptide analog; Amyloid formation; Seeding; Critical aggregation concentration

Differences in folate–protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate by Zigmund Luka; Svetlana Pakhomova; Lioudmila V. Loukachevitch; Marcia E. Newcomer; Conrad Wagner (pp. 286-291).

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate–GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate–recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.► We crystallized rat liver glycine N-methyltransferase with folate inhibitors. ► We compared structures of these complexes with that formed by recombinant protein. ► Folates establish more hydrogen bonds with native liver protein. ► This explains more efficient inhibition of liver glycine N-methyltransferase.

Keywords: Abbreviations; GNMT; glycine N-methyltransferase; AdoMet; S-adenosylmethionine; AdoHcy; S-adenosylhomocysteine; 5-CH; ₃; -H; ₄; PteGlu; ₁; 5-methyltetrahydropteroyl monoglutamate; 5-CH; ₃; -H; ₄; Pte-Glu; ₅; 5-methyltetrahydropteroyl pentaglutamateGlycine N-methyltransferase; Folate; Crystal structure; Inhibition

Copper binding traps the folded state of the SCO protein from Bacillus subtilis by Mark Lai; Katherine C. Yam; Diann Andrews; Bruce C. Hill (pp. 292-302).

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu_A center. BsSCO has been proposed to play various roles in Cu_A assembly including, the direct delivery of copper ions to the Cu_A site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu_A. BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.► apo-BsSCO behaves as a two-state protein in kinetic and equilibrium studies. ► Cu(II) binding stabilizes reduced BsSCO making unfolding extremely slow. ►Cu(II) rapidly oxidizes the two cysteines of denature BsSCO. ► BsSCO folding with Cu(II) is a competition between oxidation and folding.

Keywords: Abbreviations; BCS; bathocuproine disulfonate; EDTA; ethylenediaminetetraacetic acid; Gdn-HCl; guanidine HCl; GST; glutathione S-transferase; SCO; accessory protein required for synthesis of cytochrome; c; oxidase; BsSCO; SCO1 homolog from; Bacillus subtilis; UV-CD; circular dichroism recorded in the ultra-violet spectral region, (i.e., 180–260; nm)Copper binding; Protein folding kinetics; Protein oxidation; Cu; _A; assembly; Cytochrome; c; oxidase

Mechanism of the binding of Z–L-tryptophan and Z–L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions by Mariangela Ceruso; Nicole Howe; J. Paul G. Malthouse (pp. 303-310).

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82ppm and 182.87ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5ppm to 182.9ppm on binding to thermolysin. From pH studies we conclude that the pK_a of the inhibitor carboxylate group is lowered by at least 1.5 pK_a units when it binds to either enzyme. The signal at ~183ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-¹³C]-L-tryptophan is ≤3.71Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-¹³C]-L-tryptophan is not bound in the S₂′ subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.► The side chain of Z-Trp is not bound in the S₂′ subsite of thermolysin. ► The inhibitor carboxylate groups bind to the catalytic zinc of both enzymes. ► The inhibitor carboxylate pK_as are reduced by ≥1.5 pK_a units on enzyme binding. ► Z-Trp or Z-Phe induce tetrahedral coordination of the metal in thermolysin.

Keywords: Abbreviations; MCA; (7-methoxycoumarin-4-yl)acetyl; stromelysin-1; stromelysin-1 catalytic domain; ^83–247Thermolysin; Stromelysin-1; NMR; Amino acid; Binding; Cobalt

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c_6A by Jody M. Mason; Derek S. Bendall; Christopher J. Howe; Jonathan A.R. Worrall (pp. 311-318).

Cytochrome c_6A is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c₆ from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV ( vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c_6A from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c₆ from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c_6A and c₆ fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c_6A acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.► Equilibrium and kinetic (un)folding of two cytochrome c₆ proteins has been studied. ► The presence of a disulfide bond in cytochrome c_6A influences the global stability. ► Stopped-flow kinetics reveal the formation of an on-pathway folding intermediate. ► The disulfide bond determines the structural properties of the folding intermediate.

Keywords: Abbreviations; Cyt; cytochrome; At; Arabidopsis thaliana; Pl; Phormidium laminosum; E; _m; heme mid-point redox potential; LIP; loop insertion peptide; wt; wild-type; Amp; ampicillin; Cam; chloramphenicol; LB; lysogeny-broth; IPTG; isopropyl β-D-1-thiogalactopyranoside; GuHCl; guanidine hydrochloride; CD; Circular dichroism; NHE; normal hydrogen electrode; DTT; Dithiothreitol; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid)Cytochrome; c; _6A; Disulfide bond; Protein folding; Heme redox potential

Arabidopsis thaliana PECP1 — Enzymatic characterization and structural organization of the first plant phosphoethanolamine/phosphocholine phosphatase by Anett May; Michael Spinka; Kock Margret Köck (pp. 319-325).

Maintenance of cellular phosphate homeostasis is crucial for primary and energy metabolism. In plants, low exogenous phosphate availability activates adaptive responses that include the immediate liberation of Pi from phosphorylated metabolites by yet uncharacterized intracellular phosphatases. Based on transcriptional analyses, the Arabidopsis thaliana gene At1g17710, a member of the HAD (Haloacid Dehalogenase) superfamily, was one of the most promising candidates. Here, we show by recombinant protein production and analysis of purified protein that the gene At1g17710 encodes a phosphoethanolamine/phosphocholine phosphatase (EC 3.1.3.75). Thus, the gene product was termed AtPECP1. The present study demonstrates that the Mg2⁺-dependent enzyme exhibits pronounced specificity for both substrates. The enzyme displays a broad pH optimum ranging from pH 6 to pH 8. Comparison of K_m values indicates a slightly higher affinity for phosphocholine (0.44mM) than for phosphoethanolamine (1.16mM). The catalytic efficiency, however, is markedly higher for phosphoethanolamine than for phosphocholine being 1.06×10⁴M−¹s−¹ and 2.34×10³M−¹s−¹, respectively. Size exclusion chromatography, native gel electrophoresis and SAXS experiments with recombinant protein clearly point to a rapid monomer–dimer equilibrium of protein subunits. Given its established substrate specificity the enzyme is likely to be involved in the liberation of inorganic phosphate from intracellular sources and is especially in demand under phosphate-deprived conditions.► We identified a cytosolic enzyme function so far unknown in plants. ► The phosphatase AtPECP1 is highly specific for PCho and PEA hydrolysis. ► AtPECP1 is active in a rapid monomer-dimer equilibrium of protein subunits. ► Phosphate deprivation activates the AtPECP1gene. ► AtPECP1 participates in a phospholipid degradation pathway during Pi stress.

Keywords: HAD superfamily; Phosphatase; Phosphate starvation; PECP1; Phosphocholine; Phosphoethanolamine

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: Critical contribution of kringle-4 revealed by the mutagenesis approaches by Kishore K. Joshi; Jagpreet S. Nanda; Prakash Kumar; Girish Sahni (pp. 326-333).

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies have indicated a major role of long-range protein–protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1–K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3–K5CD (K3K4K5CD), K2–K5CD (K2K3K4K5CD) and K1–K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1–2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the ‘addition’ of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the “long-range” enzyme–substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme–substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.► Importance of kringle domains 4 and 5 in substrate plasminogen activation is shown. ► Selective kringle truncations show increase of nearly two orders of magnitude in catalysis. ► Kringle-swapping experiments were done between PG kringles and those of tPA and uPA. ► A subtle conformational trait in the substrate kringles appears to be decisive during catalysis by SK.HPN. ► This study reveals a novel, substrate-driven mechanism of action for streptokinase.

Keywords: Abbreviations; Glu-PG; K1–K5CD (closed-conformer); Lys-PG; K1–K5CD (opened-conformer); K2–K5CD; K2K3K4K5CD; K3–K5CD; K3K4K5CD; K4K5CD; kringle 4 and kringle 5 conjugated with CD; K5CD; kringle 5 conjugated with CD; CD; catalytic domain of plasminogen; μPG; microplasminogen (catalytic domain of plasminogen); μPN; microplasmin (active form of catalytic domain of plasminogen); DEAE-Sepharose; diethylaminoethyl-Sepharose; EACA; ε-aminocaproic acid; HPG; human plasminogen; HPN; human plasmin; IBs; inclusion bodies; IPTG; isopropyl-1-thio-β-; d; -galactopyranoside; k; _cat; rate of catalysis at substrate saturation; K; _m; Michaelis–Menton constant; NPGB; p-nitrophenyl p-guanidinobenzoate; PAGE; polyacrylamide gel electrophoresis; SDS; sodium dodecyl sulfate; STI; soybean trypsin inhibitor; SK; streptokinase; SAK; staphylokinase; SUPA; Streptococcus uberis; plasminogen activator; TPA; tissue plasminogen activator; uPA; urokinase plasminogen activatorFibrinolytic system; Pichia pastoris; Plasminogen; Plasminogen activator; Streptokinase; Catalysis

Binding of the plant hormone kinetin in the active site of Mistletoe Lectin I from Viscum album by Malecki Piotr H. Małecki; Wojciech Rypniewski; Szymanski Maciej Szymański; Jan Barciszewski; Arne Meyer (pp. 334-338).

The crystal structure of the ribosome inhibiting protein Mistletoe Lectin I (ML-I) derived from the European mistletoe, Viscum album, in complex with kinetin has been refined at 2.7Å resolution. Suitably large crystals of ML-I were obtained applying the counter diffusion method using the Gel Tube R Crystallization Kit (GT-R) on board the Russian Service Module on the international space station ISS within the GCF mission No. 6, arranged by the Japanese aerospace exploration agency (JAXA). Hexagonal bi-pyramidal crystals were grown during three months under microgravity. Before data collection the crystals were soaked in a saturated solution of kinetin and diffraction data to 2.7Å were collected using synchrotron radiation and cryogenic techniques. The atomic model was refined and revealed a single kinetin molecule in the ribosome inactivation site of ML-I. The complex demonstrates the feasibility of mistletoe to bind plant hormones out of the host regulation system as part of a self protection mechanism.► The kinetin molecule binds in the active side of lectin from European mistletoe. ► Different orientation of the ligand purine group compared to ATP in previous studies. ► Comparing the complex to unliganded structures, active site shows ‘open’ conformation.

Keywords: Microgravity; Ribosome inactivation; Cytokinin; Active site; Kinetin

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold by Francisco J. Fernandez; Dominique de Vries; Pena-Soler Esther Peña-Soler; Miquel Coll; Philipp Christen; Heinz Gehring; M. Cristina Vega (pp. 339-349).

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio ofl-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination ofl-cysteine sulfinate increased to 0.5s−¹ (from 0.05s−¹ in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510s−¹ to 0.05s−¹. Similarly, k_cat′ for β-decarboxylation ofl-aspartate increased from<0.0001s−¹ to 0.07s−¹, whereas k_cat′ for transamination was reduced from 530s−¹ to 0.13s−¹.l-Aspartate aminotransferase had thus been converted into anl-cysteine sulfinate desulfinase that catalyzes transamination andl-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineeredl-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme–substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme–substrate intermediates by bulk water.► We engineer aspartate aminotransferase to change substrate and reaction specificity. ► Substitutions (I33Q/Y214Q/R280Y) generate a cysteine sulfinate desulfinase. ► Desulfinase to transaminase activity fold enhancement in the mutant enzyme is 10⁵. ► X-ray structures of engineered enzyme in PLP, PMP and alanyl-PLP forms solved. ► Mutated residues are present in naturally occurring desulfinases and decarboxylases.

Keywords: Abbreviations; Ec; AspAT; Escherichia coli; aspartate aminotransferase; PLP; pyridoxal 5′-phosphate; PMP; pyridoxamine 5′-phosphateAspartate aminotransferase; Pyridoxal 5′-phosphate; Protein engineering

Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase by Zahra Amini-Bayat; Saman Hosseinkhani; Rahim Jafari; Khosro Khajeh (pp. 350-358).

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D⁴⁷⁴ and K⁴⁴⁵ accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.► Two mutations in the most flexible region of firefly luciferase were designed. ► D476N mutation doesn't have any significant effect on thermostability. ► D474K mutation destabilized protein. ► Flexibility analysis demonstrates that D474K became more flexible than native.

Keywords: Abbreviations; ATP; Adenosine triphosphate; ANS; 1-Anilino-8-naphthalene sulfonate; CD; Circular dichroism; Ni-NTA; Nickel nitrilotriacetic acid; Ppy; Photinus pyralis; Trp; TryptophanBioluminescence; Luciferase; Thermostability; Flexibility; B-factor

Structural characterization of the RNA chaperone Hfq from the nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1 by Marco Antonio Seiki Kadowaki; Jorge Iulek; João Alexandre Ribeiro Gonçalves Barbosa; Fábio de Oliveira Pedrosa; Emanuel Maltempi de Souza; Leda Satie Chubatsu; Rose Adele Monteiro; Marco Aurélio Schüler de Oliveira; Maria Berenice Reynaud Steffens (pp. 359-365).

The RNA chaperone Hfq is a homohexamer protein identified as an E. coli host factor involved in phage Qβ replication and it is an important posttranscriptional regulator of several types of RNA, affecting a plethora of bacterial functions. Although twenty Hfq crystal structures have already been reported in the Protein Data Bank (PDB), new insights into these protein structures can still be discussed. In this work, the structure of Hfq from the β-proteobacterium Herbaspirillum seropedicae, a diazotroph associated with economically important agricultural crops, was determined by X-ray crystallography and small-angle X-ray scattering (SAXS). Biochemical assays such as exclusion chromatography and RNA-binding by the electrophoretic shift assay (EMSA) confirmed that the purified protein is homogeneous and active. The crystal structure revealed a conserved Sm topology, composed of one N-terminal α-helix followed by five twisted β-strands, and a novel π-π stacking intra-subunit interaction of two histidine residues, absent in other Hfq proteins. Moreover, the calculated ab initio envelope based on small-angle X-ray scattering (SAXS) data agreed with the Hfq crystal structure, suggesting that the protein has the same folding structure in solution.► The first Hfq structure from a Gram negative diazotrophic bacterium. ► A novel π-π staking intra-subunit interaction of two histidine residues was revealed. ► The SAXS envelope suggests that the protein has the same folding in solution.

Keywords: Hfq; Herbaspirillum seropedicae; Crystal structure; SAXS

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin–myosin interaction during the ATPase cycle by Nikita A. Rysev; Olga E. Karpicheva; Charles S. Redwood; Yurii S. Borovikov (pp. 366-373).

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin–myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.

Keywords: α-Tropomyosin; Hypertrophy cardiomyopathy; Ghost muscle fiber; ATPase cycle; Polarized fluorescence

Proteomic profiling of hempseed proteins from Cheungsam by Seul-Ki Park; Jong-Bok Seo; Mi-Young Lee (pp. 374-382).

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp ( Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3–10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.► We conducted proteomic profiling of hempseed proteins from industrial hemp, Cheungsam. ► The unique 168 protein spots were identified using LC-MS/MS and searches of protein databases. ► The proteins were categorized based on their functional roles.

Keywords: Abbreviations; ACN; acetonitrile; DTE; dithioerythreitol; TCA; trichloroacetic acid; CHAPS; 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; TBP; tributylphosphine; IPG; immobilized pH gradient; 2-DE; two-dimensional gel electrophoresisCheungsam; Cannabis sativa; L.; Hempseed; Proteome; Profiling

Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin by Inés Burgos; Sergio A. Dassie; Marcos A. Villarreal; Gerardo D. Fidelio (pp. 383-391).

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein–protein interaction employing β-lactoglobulin A from bovine milk at pH=6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin ( N₂) reversibly dissociates into monomeric units ( N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature: N₂⇌2 N⇌2 D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.►We studied the thermodynamics of dissociation of dimeric β-lactoglobulin. ►Differential scanning calorimetry data was analyzed with a thermodynamic model. ►The parameters of dimerization and unfolding were obtained. ►Structural changes upon dissociation were detected with infrared spectroscopy.

Keywords: Abbreviations; N; native state; D; unfolded state; ASA; accessible surface area; PPIs; protein-protein interactions; Δ; H; _vH; van't Hoff enthalpy change; Δ; H; _cal; calorimetric enthalpy change; Δ; H; _D; unfolding enthalpy change; Δ; H; _n; association enthalpy change; n; oligomerization number; β-LG; β-lactoglobulin; ΔC; _PD; ^∘; unfolding heat capacity change evaluated at the reference temperature; ΔC; _Pn; ^∘; association heat capacity change evaluated at the reference temperature; ΔC; ′; _PD; unfolding heat capacity change temperature dependence; ΔC; ′; _Pn; association heat capacity change temperature dependence; rms; root mean square deviationOligomeric protein; Protein stability; β-lactoglobulin; Differential scanning calorimetry; Fourier-transformed infrared spectroscopy; Thermodynamic model

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803 by Sang Gon Kim; Jung-Sung Chung; R. Bryan Sutton; Jong-Sun Lee; Lopez-Maury Luis López-Maury; Sang Yeol Lee; Francisco J. Florencio; Teresa Lin; Masoud Zabet-Moghaddam; Matthew J. Wood; Kamakshi Nayak; Vivek Madem; Jatindra N. Tripathy; Sung-Kun Kim; David B. Knaff (pp. 392-403).

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation–reduction midpoint potential (E_m) value of −165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of −220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.Display Omitted► Cysteines on SynArsC and GrxA play essential roles in the reduction of arsenate. ► Understanding electron transfer between SynArsC and GrxA. ► Providing insights into the mechanism of the reaction catalyzed by SynArsC.

Keywords: Abbreviations; DTNB; 5,5′-dithiobis(2-nitorbenzoic acid); DTT; _red; reduced dithiothreitol; DTT; _ox; oxidized dithiothreitol; Grx; glutaredoxin; GSH; reduced glutathione; GSSG; oxidized glutathione; MALDI-TOF; Matrix Assisted Laser Desorption/Ionization-Time of Flight; mal-PEG; methoxy-polyethyleneglycol maleimide; mBBr; monobromobimane; RMSD; root mean square deviation; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; TCA; trichloroacetic acidArsenate reductase; Glutaredoxin; Synechocystis; Redox reactions; Dithiol–disulfide couples

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BBA - Proteins and Proteomics (v.1824, #2)