Check out our New Publishers' Select for Free Articles

BBA - Proteins and Proteomics (v.1764, #2)

Editorial Board (pp. ii).

Comparative ligand-binding analysis of ten human lipocalins by Daniel A. Breustedt; Dorian L. Sch�nfeld; Arne Skerra (pp. 161-173).

At least ten different lipocalins occur in the human body: retinol-binding protein (RBP), α₁-acid glycoprotein, α₁-microglobulin, apolipoprotein D, β-trace protein, complement component 8γ, glycodelin, neutrophil gelatinase-associated lipocalin, odorant-binding protein, and tear lipocalin. Although many of these lipocalins seem to play an important physiological role, their precise biological function is not always clear. Especially the interpretation of their diverse ligand-binding activities has been hampered by the fact that the natural lipocalins were prepared from different sources and with varying purity. Here we present a generic expression and purification strategy for the recombinant lipocalins, which is based on secretion into the periplasm of E. coli, where disulphide bonds are readily formed, followed by affinity purification via the Strep-tag II and gel filtration. The ten human lipocalins were successfully prepared and their ligand-binding activities were compared via fluorescence titration with a set of typical ligands: retinol, retinoic acid (RA), 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid (DAUDA), and 8-anilino-1-naphtalene-sulfonic acid (ANS). As result, merely two lipocalins, RBP and β-trace, revealed high affinities both for retinol and for RA, which probably reflects a specialized physiological function in retinoid complexation. Surprisingly, the strongest retinol affinity was detected for apolipoprotein D, whereas this lipocalin exhibits much weaker binding activity for retinoic acid. Binding studies with the two spectroscopic probes DAUDA and ANS revealed mixed patterns, which demonstrates that the affinity for lipophilic substances varies considerably among human lipocalins. Notably, RBP with its perfectly moulded retinol-binding site did not show any detectable binding activity for both compounds. Hence, our recombinant expression and purification system should be useful for further structural and functional studies of lipocalins from human origin and beyond.

Keywords: Abbreviations; AGP; α; ₁; -acid glycoprotein; α; ₁; m; α; ₁; -microglobulin; ANS; 8-anilino-1-naphtalene-sulfonic acid; ApoD; apolipoprotein D; BBP; bilin-binding protein; CNS; central nervous system; C8γ; complement component 8γ; DAUDA; 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid; GLY; glycodelin; NGAL; neutrophil gelatinase-associated lipocalin; OBP; odorant-binding protein; PGDS; prostaglandin D synthase (β-trace); RA; all–trans; retinoic acid; RBP; plasma retinol-binding protein; rfu; relative fluorescence units; Tlc; tear lipocalinANS; DAUDA; E. coli; secretion; Fluorescent probe; Retinoic acid; Retinol; Retinol-binding protein; Strep; -tag

Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2 by Mar�a E. Primo; Mauricio P. Sica; Valeria A. Risso; Edgardo Poskus; Mario R. Erm�cora (pp. 174-181).

The receptor protein tyrosine phosphatase superfamily (RPTP) includes proteins with a single transmembrane, one or more intracellular phosphatase, and a variety of extracellular domains. The 106-kDa insulinoma-associated protein (IA-2, ICA512) receptor is unique among RPTP members because: (a) it has a single, phosphatase-like intracellular domain identified as one of the most prominent self antigens in autoimmune diabetes; (b) its extracellular region bears no sequence similarity to known domains; (c) it is present in the membrane of secretory granules in neurons and pancreatic β-cells where it suffers a complex processing; and (d) it has very poorly understood biological properties. In this work, we describe the expression, purification, and physicochemical characterization of residues 449–576 of IA-2 (IA-2ec^449–576). Judging from CD, fluorescence, hydrodynamic, and thermal unfolding analyses, this fragment forms an autonomously folding unit with tight packing and well-defined secondary and tertiary structure. CD analysis suggests that about 25% of IA-2ec^449–576 residues are α-helical, whereas about the same amount are in β-sheet structure. The availability of soluble and folded IA-2ec^449–576 is a step forward toward the characterization of a part of IA-2 at atomic detail, which may provide new insight in the biology of diabetes, the neurotransmission process, and the dynamic of secretory granules.

Keywords: IA-2; ICA-512; Diabetes; Tyrosine phosphatase

Thermodynamic characterization of interactions between p27^Kip1 and activated and non-activated Cdk2: Intrinsically unstructured proteins as thermodynamic tethers by Prentice Bowman; Charles A. Galea; Eilyn Lacy; Richard W. Kriwacki (pp. 182-189).

The cyclin-dependent kinase inhibitor (CKI) p27^Kip1 plays a critical role in cell cycle regulation by binding and inhibiting (or activating) various cyclin-dependent kinase (Cdk)/cyclin complexes. Thermal denaturation monitored by circular dichroism (CD) and isothermal titration calorimetry (ITC) were used to determine the relative stabilities and affinities of p27-KID (p27 kinase inhibitory domain) complexes with activated Cdk2 (phosphorylated at Thr160; P-Cdk2) and non-activated forms of Cdk2 and/or cyclin A. Phosphorylation of residue Thr160 only slightly increases the thermal stability of Cdk2, and its binary complexes with cyclin A and p27-KID. The p27-KID/P-Cdk2/cyclin A or p27-KID/Cdk2/cyclin A ternary complexes exhibited significantly higher thermal stabilities compared to the binary complexes (P-Cdk2/cyclin A or Cdk2/cyclin A). Differences in T_m values between the binary and ternary complexes with P-Cdk2 and Cdk2 were +25.9 and +20.4 °C, respectively. These results indicate that the ternary complex with phosphorylated Cdk2 is stabilized to a larger extent than the non-phosphorylated complex. The free energy of association (Δ G_A) for formation of the two ternary complexes was more favorable than for the binary complexes, indicating that a significantly smaller population of free components existed when all three components were present. These data indicate that p27-KID, which is intrinsically disordered in solution, acts as a thermodynamic tether when bound within the ternary complexes. It is proposed that thermodynamic tethering may be a general phenomena associated with intrinsically unstructured proteins (IUPs) which often function by binding to multiple partners in multi-protein assemblies.

Keywords: p27-KID; Cell cycle; Cyclin-dependent kinase inhibitor; Isothermal titration calorimetry; Thermal denaturation; Intrinsically unstructured protein

Reversible inactivation of alkaline phosphatase from Atlantic cod ( Gadus morhua) in urea by Bjarni �sgeirsson; Katr�n Gu�j�nsd�ttir (pp. 190-198).

Alkaline phosphatase (AP) from Atlantic cod ( Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6–8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity.

Keywords: Abbreviations; AP; Alkaline phosphatase (EC 3.1.3.1); ANS; 8-Anilino-1-naphtalene sulfonic acid; GdmCl; Guanidine hydrochloride; PAR; 4-(2-pyridylazo)resorcinolCold-adaptation; Psychrophilic; Dimer; Metalloenzyme; Folding; Stability

The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: Inactivation by 2′,3′-dialdehyde ATP and mutational analyses by Travis J. MacLeod; Faylene A. Lunn; Stephen L. Bearne (pp. 199-210).

Cytidine 5′-triphosphate synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH₃ orl-glutamine as the source of nitrogen. To identify the location of the ATP-binding site within the primary structure of E. coli CTPS, we used the affinity label 2′,3′-dialdehyde adenosine 5′-triphosphate (oATP). oATP irreversibly inactivated CTPS in a first-order, time-dependent manner while ATP protected the enzyme from inactivation. In the presence of 10 mM UTP, the values of k_inact and K_I were 0.054±0.001 min⁻¹ and 3.36±0.02 mM, respectively. CTPS was labeled using (2,8-³H)oATP and subsequently subjected to trypsin-catalyzed proteolysis. The tryptic peptides were separated using reversed-phase HPLC, and two peptides were identified using N-terminal sequencing (S(492)GDDQLVEIIEVPNH(506) and Y(298)IELPDAY(K(306)) in a 5:1 ratio). The latter suggested that Lys 306 had been modified by oATP. Replacement of Lys 306 by alanine reduced the rate of oATP-dependent inactivation ( k_inact=0.0058±0.0005 min⁻¹, K_I=3.7±1.3 mM) and reduced the apparent affinity of CTPS for both ATP and UTP by approximately 2-fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation was also reduced 2-fold while near wild-type activity was observed when NH₃ was the substrate. These findings suggest that Lys 306 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between the ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain. Replacement of the nearby, fully conserved Lys 297 by alanine did not affect NH₃-dependent CTP formation, relative to wild-type CTPS, but reduced k_cat for the glutaminase activity 78-fold. Our findings suggest that the conformational change associated with binding ATP may be transmitted through the L10-α11 structural unit (residues 297–312) and thereby mediate effects on the glutaminase activity of CTPS.

Keywords: Abbreviations; CTPS; cytidine 5′-triphosphate synthase; GAT; glutamine amide transfer; oATP; 2′,3′-dialdehyde adenosine 5′-triphosphate; PTH; phenylthiohydantoin; RP-HPLC; reversed-phase high performance liquid chromatographyCTP synthase; ATP-binding site; Amidotransferase; Affinity label; 2′,3′-dialdehyde ATP; Inactivation; E. coli; GTP-dependent activation

β-lactoglobulin under high pressure studied by small-angle neutron scattering by C. Loupiac; M. Bonetti; S. Pin; P. Calmettes (pp. 211-216).

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of β-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.

Keywords: β-lactoglobulin; High pressure; Molten globule; Aggregation; Small-angle neutron scattering

Effect of nucleotide on interaction of the 567–578 segment of myosin heavy chain with actin by Brian M. Martin; Emilia Karczewska; Barbara Pliszka (pp. 217-222).

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567–578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF₄, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567–578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567–578 loop that can be cross-linked to actin in the presence of MgADP.AlF₄ is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567–578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.

Keywords: Abbreviations; S1; myosin subfragment 1; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDC; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; IAEDANS; N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamineActin–myosin interaction; Subfragment 1; Chemical cross-linking

Kinetic characterization of Synechocystis sp. PCC6803 1-deoxy-d-xylulose 5-phosphate reductoisomerase mutants by Roberta P.M. Fernandes; Philip J. Proteau (pp. 223-229).

The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-d-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.

Keywords: Abbreviations; BSA; bovine serum albumin; DXR; 1-deoxy-; d; -xylulose 5-phosphate reductoisomerase; DXP; 1-deoxy-; d; -xylulose 5-phosphate; MEP; 2-; C; -methyl-; d; -erythritol 4-phosphateDeoxyxylulose 5-phosphate; Isoprenoid; Mutagenesis; Reductoisomerase; Synechocystis

Ionization state of pyridoxal 5′-phosphate ind-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by³¹P NMR spectroscopy by Klaus D. Schnackerz; John Keller; Robert S. Phillips; Michael D. Toney (pp. 230-238).

The³¹P NMR spectroscopy of three pyridoxal 5′-phosphate-dependent enzymes, monomericd-serine dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied.³¹P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by ∼3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The³¹P NMR signal of the phosphate group of pyridoxal 5′-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. Ford-serine dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from³¹P NMR data.

Keywords: Abbreviations; DGD; dialkylglycine decarboxylase; DGD-K; ⁺; DGD-Na; ⁺; , DGD-Li; ⁺; , DGD-Rb; ⁺; , DGD saturated with potassium, sodium, lithium or rubidium ions, respectively; DSD; d; -serine dehydratase; TPL; tyrosine phenol-lyase; PLP; pyridoxal 5′-phosphate; PMP; pyridoxamine 5′-phosphate; PPL-Asp; phosphopyridoxyl aspartate; Mes; 2-(N-morpholino)ethane sulfonic acid; Hepes; N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; Bis-Tris; bis-Tris propane; AIB; α-aminoisobutyrated; -serine dehydratase; Dialkylglycine decarboxylase; Tyrosine phenol-lyase; Monovalent cations; 31P chemical shift; Ppyridoxal 5'-phosphate

Biochemical characterization of a truncated penta-EF-hand Ca²⁺ binding protein from maize by Jennifer K. Barry; David A. Selinger; Cunxi Wang; Odd-Arne Olsen; A. Gururaj Rao (pp. 239-245).

Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca²⁺ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.

Keywords: Abbreviations; ALG-2; apoptosis-linked gene 2; PEF; penta-EF-hand; PPC; Plant PEF type C; TNS; 2-p-toluidinylnaphthalene sulfonate ZmPEF-C, PPC gene in MaizeCalcium binding protein; Penta-EF-hand protein; Calcium sensing

Solution¹H NMR investigation of Zn²⁺ and Cd²⁺ binding to amyloid-beta peptide (Aβ) of Alzheimer's disease by Christopher D. Syme; John H. Viles (pp. 246-256).

Elevated levels of zinc²⁺ and copper²⁺ are found chelated to the amyloid-beta-peptide (Aβ) in isolated senile plaque cores of Alzheimer's disease (AD) patients. However, the precise residues involved in Zn²⁺ ligation are yet to be established. We have used¹H NMR and CD to probe the binding of Zn²⁺ to Aβ(1–28). Zinc binding to Aβ causes a number of¹H NMR resonances to exhibit intermediate exchange broadening upon Zn²⁺ addition, signals in slow and fast exchange are also observed. In addition, there is a general loss of signal for all resonances with Zn²⁺ addition, suggestive of the formation of high molecular weight polymeric species. Perturbations in specific¹H NMR resonances between residues 6 and 14, and analysis of various Aβ analogues in which each of the three His residues have been replaced by alanine, indicates that His6, His13 and His14 residues are implicated in Zn-Aβ binding. Complementary studies with Cd²⁺ ions cause perturbations to¹H NMR spectra that are strikingly similar to that observed for Zn²⁺. Binding monitored at Val12 indicates a 1:1 stoichiometry with Aβ for both Zn²⁺ and Cd²⁺ ions. Circular Dichroism (CD) studies in the far-UV indicate quite minimal ordering of the main-chain with Zn²⁺ or Cd²⁺ addition. Changes in the far-UV are quite different from that obtained with Cu²⁺ additions indicating that Zn²⁺ coordination is distinct from that of Cu²⁺ ions. Taken together, these observations seem to suggest that Zn²⁺ coordination is dominated by inter-molecular coordination and the formation of polymeric species.

Keywords: Abbreviations; Aβ; amyloid-β peptide; AD; Alzheimer's disease; CD; circular dichroism; CSF; cerebrospinal fluid; NMR; Nuclear Magnetic ResonanceZinc; Spectroscopy; Structure; CD; Aggregation

Study on the binding of Arsenazo-TB to human serum albumin by Rayleigh light scattering technique and FT-IR by Lijun Dong; Xingguo Chen; Zhide Hu (pp. 257-262).

The interaction between Arsenazo-TB and human serum albumin (HSA) was studied by Rayleigh light scattering (RLS) technique and Fourier transformed IR (FT-IR). The binding parameters of Arsenazo-TB with HSA were studied at different temperature of 288, 298, 308, 318 K under the optimum conditions. It is indicated by the Scatchard plots that the binding constant K decreased from 5.03×10⁷ to 7.13×10⁶ and the maximum binding number N reduced from 53 to 36 with the increasing of the temperature. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The free energy change Δ G⁰, the enthalpy change Δ H⁰ and the entropy change Δ S⁰ of 288 K were calculated to be −42.46 kJ/mol, −49.17 kJ/mol and 318.15 J/mol K, respectively. The alterations of protein secondary structure in the presence of Arsenazo-TB in aqueous solution were quantitatively calculated from FT-IR spectroscopy with reductions of α-helix from 57% to 40% and with increases of β-sheet from 36% to 39%, β-turn from 7% to 21%.

Keywords: Arsenazo-TB; Human serum albumin; Rayleigh light scattering; Fourier transformed IR

Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4 by Vladim�r Puchart; Marie-Claude Gari�py; Fran�ois Shareck; Claude Dupont (pp. 263-274).

Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.

Keywords: Abbreviations; AxeA; Streptomyces lividans; acetylxylan esterase A; AxeA; _tr; truncated AxeA; CE4; carbohydrate esterase family 4; PdaA; Bacillus subtilis; 168 peptidoglycan; N; -acetylmuramic acid deacetylase ACarbohydrate esterase family 4; Acetylxylan esterase; Streptomyces lividans; Aspartic deacetylase

The activity of barley α-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase by Nathalie Juge; Jane N�hr; Marie-Fran�oise Le Gal-Co�ffet; Birte Kramh�ft; Caroline S.M. Furniss; V�ronique Planchot; David B. Archer; Gary Williamson; Birte Svensson (pp. 275-284).

High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley α-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg×L⁻¹ and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1–5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U×nmol⁻¹ and K_d=0.13 mg×mL⁻¹, respectively, were both improved with respect to the values 0.015 U×nmol⁻¹ and 0.67 mg×mL⁻¹ for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.

Keywords: Abbreviations; ACMM/N-C; Aspergillus niger; complete medium with maltose and additional nitrogen buffered with citrate; AMY1 and AMY2; barley α-amylase isozymes 1 and 2; AMY1-SBD; recombinant AMY1-SBD; AMY1-SBD; cDNA encoding the residues 1–414 of AMY1 fused to residues 471–616 of GA-I; BNPG; ₅; blocked p-nitrophenyl α-; d; -maltopentaoside; BNPG; ₇; blocked 4-nitrophenyl α-; d; -maltohepatoside; bp; base pair; CBM20; carbohydrate binding module family 20; CGTase; cyclodextrin glucosyltransferase; Cl-PNPG; ₇; 2-chloro-4-nitrophenol β-; d; -maltohepatoside; DMA; degree of multiple attack; DMSO; dimethylsulfoxide; DP; degree of polymerisation; GA; glucoamylase; GA-I; A. niger; glucoamylase 1 (aa 1–616); GA-II (aa 1–512); as GA-I but lacking SBD; GH13, GH14, GH15; glycoside hydrolase families 13, 14, 15; MTH; maltotetraose-forming hydrolase; rAMY1; recombinant AMY1 produced in; A. niger; SBD; starch binding domain from GA-I (residues 509–616)Glycoside hydrolase family 13; Starch binding domain fusion; Carbohydrate binding module family 20; O; -glycosylated linker region; Starch granule hydrolysis; Aspergillus niger

Structural changes accompanying human serum albumin's binding of fatty acids are concerted by Yunnan Fang; Grace C. Tong; Gary E. Means (pp. 285-291).

Long chain fatty acids (LCFAs), a major source of cellular energy, are solubilized and transported in the blood by binding to serum albumin. Changes in human serum albumin's (HSA's) UV absorption and characteristic reactivity with pyridoxal-5′-phosphate appear to reflect a concerted change in its structure upon binding five equivalents of myristate. Isothermal titrations with myristate and other LCFA anions are also consistent with the presence of five strong, interacting, binding sites. Although HSA is usually thought to have many independent LCFA anion binding sites, just five interacting sites appear to account for the changes in structure that accompany its binding of myristate.

Keywords: Abbreviations; HSA; human serum albumin; LCFA; long chain fatty acid; PLP; pyridoxal-5′-phosphate; ITC; isothermal titration calorimetryHuman serum albumin; Cooperative binding; Regulation of fatty acid level; Protein conformation change; Isothermal titration calorimetry; Pyridoxal phosphate reactivity

Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis by Montserrat And�jar-S�nchez; Sergio Mart�nez-Rodr�guez; Francisco Javier Las Heras-V�zquez; Josefa Mar�a Clemente-Jim�nez; Felipe Rodr�guez-Vico; Vicente Jara-P�rez (pp. 292-298).

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecificd-carbamoylase guarantee the total conversion fromd,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pured-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, Δ G, Δ H and Δ S for the inhibitorsl- andd-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.

Keywords: Abbreviations; ITC; Isothermal titration calorimetry; MTEH; Methylthioethyl hydantoin; SmeHyuA; hydantoin racemase from; Sinorhizobium meliloti; CECT 4114; ASA; Accessible surface area; HEPES; N; -2-hydroxyethylpiperazine-; N; ′-2-ethanesulfonic acid; ACES; N; -(2-Acetamido)-2-aminoethanesulfonic acidBinding; Mutagenesis; Hydantoin racemase; Inhibition; Thermodynamic parameter

Casein kinases phosphorylate multiple residues spanning the entire hnRNP K length by Michał Mikula; Jakub Karczmarski; Artur Dzwonek; Tymon Rubel; Ewa Hennig; Michał Dadlez; Janusz M. Bujnicki; Karol Bomsztyk; Jerzy Ostrowski (pp. 299-306).

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is an RNA/DNA-binding protein involved in many processes that regulate gene expression. K protein's pleiotropic action reflects the diversity of its molecular interactions. Many of these interactions have been shown to be regulated by phosphorylation. K protein contains more than seventy potential phosphorylation sites. We used an integrated approach of mass spectrometry and computer analysis to explore patterns of K protein phosphorylation. We found that in vitro a single kinase can phosphorylate K protein on multiple sites spanning the entire length of the protein, including residues contained within the RNA/DNA-binding domains. 2-D gel electrophoresis of K protein purified from cells identified 5–8 spots. Mass spectrometry of K protein isolated from proliferating cells and from cells under oxidative stress revealed the same pattern of phosphopeptides. The structural implications of phosphorylation are discussed.

Keywords: hnRNPK; Immunoprecipitation; Casein kinases; Phosphorylation; Protein modeling; Mass spectrometry

Analysis of the kinetic mechanism of arginyl-tRNA synthetase by R. Kalervo Airas (pp. 307-319).

A kinetic analysis of the arginyl-tRNA synthetase (ArgRS) from Escherichia coli was accomplished with the goal of improving the rate equations so that they correspond more closely to the experimental results. 22 different steady-state kinetic two-ligand experiments were statistically analysed simultaneously. A mechanism and values for the ArgRS constants were found where the average error was only 6.2% and ranged from 2.5 to 11.2% in the different experiments. The mechanism included not only the normal activation and transfer reactions but also an additional step which may be a conformational change after the transfer reaction but before the dissociation of the product Arg-tRNA from the enzyme. The forward rate constants in these four steps were low, 8.3–27 s⁻¹, but the reverse rate constants of the activation and transfer reactions were considerably higher (230 and 161 s⁻¹). Therefore, in the presence of even low concentrations of PP_i and AMP, the rate limitation occurs at the late steps of the total reaction. AMP increases the rate of the ATP-PP_i exchange reaction due to the high reverse rate in the transfer reaction. The rate equation obtained was used to calculate the steady-state enzyme intermediate concentrations and rates between the intermediates. Three different Mg²⁺ binding sites were required to describe the Mg²⁺ dependence. One of them was the normal binding to ATP and the others to tRNA or enzyme. The measured Mg²⁺ dependence of the apparent equilibrium constant of the ArgRS reaction was consistent with the Mg²⁺ dependences of the reaction rates on the rate equation. Chloride inhibits the ArgRS reaction, 160 mM KCl caused a 50% inhibition if the ionic strength was kept constant with K-acetate. KCl strongly affected the K_m^app (tRNA) value. A difference was detected in the progress curves between the aminoacylation and ATP-PP_i exchange rates. When all free tRNA^Arg had been used from the reaction mixture, the aminoacylation reaction stopped, but the ATP-PP_i exchange continued at a lowered rate.

Keywords: Abbreviation; ArgRS; arginyl-tRNA synthetaseSynthetase; tRNA; Arginyl-tRNA synthetase; Magnesium; Kinetic

Biochemical characterization and preliminary X-ray crystallographic study of the domains of human ZBP1 bound to left-handed Z-DNA by Sung Chul Ha; Dong Van Quyen; Hye-Yeon Hwang; Doo-Byoung Oh; Bernard A. Brown II; Seon Min Lee; Hyun-Ju Park; Jin-Hyun Ahn; Kyeong Kyu Kim; Yang-Gyun Kim (pp. 320-323).

ZBP1 is involved in host responses against cellular stresses, including tumorigenesis and viral infection. Structurally, it harbors two copies of the Zα domain containing the Zα motif, at its N terminus. Here, we attempted to characterize the Z-DNA binding activities of two Zα domains in the human ZBP1, hZα_ZBP1 and hZβ_ZBP1, using circular dichroism (CD). Our results indicated that both hZα_ZBP1 and hZβ_ZBP1 are viable Z-DNA binders, and their binding activities are comparable to those of previously-established Zα domains. Additionally, we crystallized hZβ_ZBP1 in a complex with Z-DNA, d(TCGCGCG)₂. The crystal diffracted to 1.45 Å, and belongs to the P2₁2₁2₁ space group, with the unit-cell parameters: a=29.53 Å, b=58.25 Å, and c=88.61 Å. The delineation of this structure will provide insight into the manner in which diverse Zα motifs recognize Z-DNA.

Keywords: Zα motif; ZBP1; Z-DNA; DNA binding; Circular dichroism; Crystallization

Protein preparation, crystallization and preliminary X-ray crystallographic analysis of Smu.1475c from caries pathogen Streptococcus mutans by Yan-Feng Zhou; Wei Mi; Lanfen Li; Xiaoyan Zhang; Yu-He Liang; Xiao-Dong Su; Shicheng Wei (pp. 324-326).

The gene smu.1475c encodes a putative protein of 211 residues in Streptococcus mutans, a primary pathogen for human dental caries. In this work, smu.1475c was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.1475c protein was purified to homogeneity in a two-step procedure of Ni²⁺ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor-diffusion method and diffracted to 2.7 � resolution. The crystal belongs to orthorhombic space group P2₁2₁2₁ with cell dimension of a=68.3 �, b=105.9 �, c=136.2 �. The asymmetric unit is expected to contain four molecules with solvent content of 49.4%.

Keywords: Streptococcus mutans; Dental caries; Smu.1475c; Protein crystallography

Corrigendum to: “Crystallization and preliminary X-ray crystallographic analysis of extracellular giant hemoglobin from pogonophoran Oligobrachia mashikoi�? [Biochim. Biophys. Acta 1750 (2005) 173–176] by Nobutaka Numoto; Taro Nakagawa; Akiko Kita; Yuichi Sasayama; Yoshihiro Fukumori; Kunio Miki (pp. 327-327).

Sections

Personal tools

BBA - Proteins and Proteomics (v.1764, #2)

Sections

Personal tools

Local resources

BBA - Proteins and Proteomics (v.1764, #2)