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BBA - Proteins and Proteomics (v.1784, #3)
Amidohydrolases of the reductive pyrimidine catabolic pathway by Klaus D. Schnackerz; Doreen Dobritzsch (pp. 431-444).
In the reductive pyrimidine catabolic pathway uracil and thymine are converted to β-alanine and β-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyrate (β-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the β-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. β-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.
Keywords: Abbreviations; βAS; β-alanine synthase; Dm; βAS; β-alanine synthase from; Drosophila melanogaster; Sk; βAS; β-alanine synthase from; Saccharomyces kluyveri; DHPase; dihydropyrimidine amidohydrolase; Dd; DHPase; dihydropyrimidine amidohydrolase from; Dictyostelium discoideum; Sk; DHPase; dihydropyrimidine amidohydrolase from; Saccharomyces kluyveri; DHU; 5,6-dihydrouracil, DHT, 5,6-dihydrothymine; NCβA; N; -carbamyl-β-alanine; Hyd; hydantoinases; DHOase; dihydroorotase; 5FU; 5-fluorouracil; SGL; stereochemistry gate loop; acy1; aminoacylase 1; SGAP; Streptomyces griseus; aminopeptidase; AAP; Aeromonas proteolytica; aminopeptidase; CPG2; Pseudomonas sp; . carboxypeptidase G2; PepT; aminotripeptidase T from; Salmonella typhimurium;; PepV; ,; peptidase V from; Lactobacillus delbrueckii; B9; DHyd and; Bs; DHyd; d; -hydantoinases from; Bacillus sp; .; Bp; DHyd; d; -hydantoinase from; Burkholderia pickettii; Ts; DHyd; d; -hydantoinase from; Thermus sp.;; Aa; Lhyd,; l; -hydantoinase from; Arthrobacter aurescensDihydropyrimidine amidohydrolase; β-Alanine synthase; Hydantoinases; Amidohydrolase superfamily; Nitrilase superfamily; Three-dimensional structure; Di-zinc proteins; Amino acid triad; β-Alanine aminotransferase
Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: The importance of the quaternary structure by Sha Wang; Wei-Feng Liu; Yong-Zhi He; Ao Zhang; Li Huang; Zhi-Yang Dong; Yong-Bin Yan (pp. 445-454).
Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.
Keywords: Abbreviations; SOD; superoxide dismutase; GdnHCl; guanidinium chloride; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; ANS; 1-aniline-8-naphthalenesulfonate; CD; circular dichroism; E; m; emission maximum wavelength of intrinsic fluorescence; SEC; size-exclusion chromatography; ROS; reactive oxygen speciesFe-superoxide dismutase; Hyperthermostable; Protein unfolding and refolding; Folding intermediate; Metalloenzyme
Investigating the biomarker potential of glycoproteins using comparative glycoprofiling — application to tissue inhibitor of metalloproteinases-1 by Morten Thaysen-Andersen; Ida B. Thøgersen; Ulrik Lademann; Hanne Offenberg; Anders M.B. Giessing; Jan J. Enghild; Hans Jørgen Nielsen; Nils Brünner; Peter Højrup (pp. 455-463).
Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.
Keywords: Abbreviations; CCC; colon cancer cell line; CRC; colorectal cancer; HILIC; hydrophilic interaction liquid chromatography; MALDI; matrix-assisted laser desorption/ionisation; MMP; matrix metalloproteinase; MS; mass spectrometry; MS/MS; tandem mass spectrometry; PCA; principal component analysis; PCC; prostate cancer cell line; PSA; prostate specific antigen; Q; quadrupole; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TIMP-1; tissue inhibitor of metalloproteinases-1; TOF; time-of-flightGlycoprofiling; Colorectal cancer; Mass spectrometry; TIMP-1; Biomarker
Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail ( Cortunix cortunix japonica) by Kehinde Onwochei Okonjo; Olugbenga S. Bello; J. Oyebamiji Babalola (pp. 464-471).
We demonstrate that 5,5′-dithiobis(2-nitrobenzoate) – DTNB – reacts with only CysF9[93]β and CysB5[23]β among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail ( Cortunix cortunix japonica). Kequ, the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH≈5.6 and pH≈9: from a mean of 7±1 to a mean of 0.016±0.003. Quantitative analyses of the Kequ data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of Krt, the equilibrium constant for ther←→t tertiary structure transition. Krt differs significantly between the two hemoglobins: 0.744±0.04 for the major, 0.401±0.01 for the minor hemoglobin. The mean p Kas of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins.
Keywords: Japanese quail hemoglobin; Tertiary conformational transition; Multiple reactive sulfhydryl group; Reaction with 5,5′-dithiobis(2-nitrobenzoate); Equilibrium constant
Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme by Bing-Rui Zhou; Zheng Zhou; Qing-Lian Hu; Jie Chen; Yi Liang (pp. 472-480).
The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.
Keywords: Abbreviations; BSA; bovine serum albumin; CR; Congo red; HSA; human serum albumin; ITC; isothermal titration calorimetry; TEM; transmission electron microscopy; ThT; thioflavin TMacromolecular crowding; Amyloid fibril; Lysozyme; Inhibition; Kinetics
Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability by Teisuke Takita; Takahiro Aono; Haruko Sakurama; Takafumi Itoh; Takumi Wada; Masashi Minoda; Kiyoshi Yasukawa; Kuniyo Inouye (pp. 481-488).
Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the kcat/ Km values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17–19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl2 at 85 °C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35–53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl2 and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca2+-binding structures.
Keywords: Abbreviations; FAGLA; N; -[3-(2-furyl)acryloyl]-glycyl-; l; -leucine amideMetalloproteinase; Molecular surface; Site-directed mutagenesis; Thermal stability; Thermolysin
Preheating induced homogeneity of the small heat shock protein from Methanococcus jannaschii by Aoneng Cao; Zheng Wang; Ping Wei; Fei Xu; Jie Cao; Luhua Lai (pp. 489-495).
Small heat shock proteins usually exhibit increased chaperone-like activity either at high temperatures or after preheating. However, the activation mechanism is still unclear. In the current study, we investigated the preheating-activation process of Mj HSP16.5, using various biophysical methods. Although Mj HSP16.5 was reported to be the most monodispersed sHSPs, we found that the newly purified Mj HSP16.5 was actually heterogeneous. 85 °C-preheating could activate Mj HSP16.5 and turn it into a more compact homogeneous species at the same time. Different cooling rates after preheating did not change the activity of Mj HSP16.5, suggesting that the 85 °C-preheated Mj HSP16.5 is in the most active and also the most stable state. These results demonstrate that the activation process of Mj HSP16.5 might accompany a refolding process.
Keywords: Abbreviations; sHSP; small heat shock protein; Mj HSP16.5; sHSP from; Methanococcus jannaschii; CS; citrate synthase; DTT; DL-dithiothreitol; SCM; single chain monellin; SEC; size exclusion chromatography; FRET; fluorescence resonance energy transfer; DLS; dynamic light scattering; AUC; analytical ultracentrifugationHeat shock protein; Chaperone; Protein folding; Homogeneity; HSP 16.5
Cloning, purification and characterization of the Caenorhabditis elegans small glutamine-rich tetratricopeptide repeat-containing protein by Liam J. Worrall; Martin A. Wear; Antony P. Page; Malcolm D. Walkinshaw (pp. 496-503).
We have cloned and expressed the putative Caenorhabditis elegans orthologue for small glutamine-rich tetratricopeptide repeat-containing protein, now assigned the gene name sgt-1 in the C. elegans genome database. Characterization of the purified protein by cross-linking, mass spectrometry and gel filtration experiments provides unambiguous evidence that SGT-1 forms homo-dimers in solution. The hydrodynamic dimensions of SGT-1 dimers in relation to their molecular weight suggest a protein with a low level of compactness and an extended conformation. Human SGT has been shown to interact with and regulate the activity of heat shock proteins Hsp70 and Hsp90 via a TPR domain mediated interaction. The SGT TPR domain (SGT-1-TPR, residues 100–226) was cloned, purified and shown by ITC and CD analysis to interact with the C-terminal peptides of Hsp70 and Hsp90 with comparable affinities although there is no evidence of a recently proposed coupled binding–folding mechanism for TPR domains.
Keywords: Small glutamine-rich tetratricopeptide repeat-containing protein; SGT; TPR; Hsp70; Hsp90; Co-chaperone
Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon by Apichai Bourchookarn; Phattara-Orn Havanapan; Visith Thongboonkerd; Chartchai Krittanai (pp. 504-511).
A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI–TOF MS and nanoLC–ESI–MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.
Keywords: Abbreviations; ER; endoplasmic reticulum; HCP; hemolymph clottable protein; hpi; hours post-infection; LO; lymphoid organ; PDI; protein disulfide isomerase; TGase; transglutaminase; YHV; yellow head virus Penaeus monodon; Proteomics; Lymphoid organ; Yellow head virus
Iodination of salicylic acid improves its binding to transthyretin by Luís Gales; Maria Rosário Almeida; Gemma Arsequell; Gregorio Valencia; Maria João Saraiva; Ana Margarida Damas (pp. 512-517).
Transthyretin (TTR) is a plasma homotetrameric protein associated with senile systemic amyloidosis and familial amyloidotic polyneuropathy. In theses cases, TTR dissociation and misfolding induces the formation of amyloidogenic intermediates that assemble into toxic oligomeric species and lead to the formation of fibrils present in amyloid deposits. The four TTR monomers associate around a central hydrophobic channel where two thyroxine molecules can bind simultaneously. In each thyroxine binding site there are three pairs of symmetry related halogen binding pockets which can accommodate the four iodine substituents of thyroxine. A number of structurally diverse small molecules that bind to the TTR channel increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis have been tested. In order to take advantage of the high propensity to interactions between iodine substituents and the TTR channel we have identified two iodinated derivatives of salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid, available commercially. We report in this paper the relative binding affinities of salicylic acid and the two iodinated derivatives and the crystal structure of TTR complexed with 3,5-diiodosalicylic acid, to elucidate the higher binding affinity of this compound towards TTR.
Keywords: Transthyretin; Amyloid; Crystal structure; Amyloid inhibitors; Familial amyloidotic polyneuropathy (FAP)
Trimeric reassembly of the globular domain of human C1q by Pascale Tacnet; Eric Chung Chee Cheong; Pierrette Goeltz; Berhane Ghebrehiwet; Gérard J. Arlaud; Xiang-Yang Liu; Claire Lesieur (pp. 518-529).
C1q is a versatile recognition protein which binds to a variety of targets and consequently triggers the classical pathway of complement. C1q is a hetero-trimer composed of three chains (A, B and C) arranged in three domains, a short N-terminal region, followed by a collagenous repeat domain that gives rise to the formation of (ABC) triple helices, each ending in a C-terminal hetero-trimeric globular domain, called gC1q, which is responsible for the recognition properties of C1q. The mechanism of the trimeric assembly of C1q and in particular the role of each domain in the process is unknown. Here, we have investigated if the gC1q domain was able to assemble into functional trimers, in vitro, in the absence of the collagenous domain, a motif known to promote obligatory trimers in other proteins. Acid-mediated gC1q protomers reassembled into functional trimers, once neutralized, indicating that it is the gC1q domain which possesses the information for trimerization. However, reassembly occurred after neutralization, only if the gC1q protomers had preserved a residual tertiary structure at the end of the acidic treatment. Thus, the collagenous domain of C1q might initialize the folding of the gC1q domain so that subsequent assembly of the entire molecule can occur.
Keywords: C1q; Assembly mechanism; Trimer; Folding; Function
Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein by Roxana E. Iacob; Zhenyong Keck; Oakley Olson; Steven K.H. Foung; Kenneth B. Tomer (pp. 530-542).
Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.
Keywords: Abbreviations; GNA; galanthus nivalis antigen, AP, alkaline phosphatase; FACS; fluorescent-activated cell sorting; FITC; fluorescein isothiocyanate; FCS; fetal calf serum; BS3; bis(sulfosuccinimidyl)suberate; GnHCl; guanidine hydrochloride; PBS; phosphate buffered saline; CRC; compact reaction columns; MALDI; matrix assisted laser desorption/ionization; ESI; electrospray ionization; TOF; time of flight; ESI-MS/MS; electrospray-tandem mass spectrometry; LC/MS/MS; liquid chromatography-tandem mass spectrometry; HPLC; high performance liquid chromatographyHepatitis C E2 glycoprotein; Human neutralizing and non-neutralizing antibodies; Limited proteolysis; Differential chemical modification; Mass spectrometry; Alanine scanning mutagenesis
Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G by Per Rogne; Gunnar Fimland; Jon Nissen-Meyer; Per Eugen Kristiansen (pp. 543-554).
The three-dimensional structures of the two peptides, lactococcin G-α (LcnG-α; contains 39 residues) and lactococcin G-β (LcnG-β, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-α has an N-terminal α-helix (residues 3–21) that contains a GxxxG helix–helix interaction motif (residues 7–11) and a less well defined C-terminal α-helix (residues 24–34), and in between (residues 18–22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-β has an N-terminal α-helix (residues 6–19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18–22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined α-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-α and -β, are amphiphilic. We postulate that LcnG-α and -β have a parallel orientation and interact through helix–helix interactions involving the first GxxxG (residues 7–11) motif in LcnG-α and the one (residues 18–22) in LcnG-β, and that they thus lie in a staggered fashion relative to each other.
Keywords: Abbreviations; AMPs; antimicrobial peptides; CD; circular dichroism; CSI; chemical shift index; DPC; dodecylphosphocholine; DSS; 2, 2-dimethyl-2-silapentane-5-sulfonate; Ent; enterocin 1071; HSQC; heteronuclear single quantum coherence; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; LAB; lactic acid bacteria; LcnG; lactococcin G; LcnQ; lactococcin Q; MALDI-TOF; matrix assisted laser disorption-time of flight; NMR; nuclear magnetic resonance; NOESY; nuclear Overhauser effect spectroscopy; TFA; trifluoroacetic acid; TFE; trifluoroethanol; TOCSY; total correlation spectroscopyLactococcin G; Antimicrobial peptide; Peptide structure; Lactic acid bacteria; NMR-spectroscopy
Modulation of the hippocampal protein machinery in voluntary and treadmill exercising rats by Wei-Qiang Chen; Wei-Fei Diao; Andrus Viidik; Monika Skalicky; Harald Höger; Gert Lubec (pp. 555-562).
Information about protein expression studies in the brain of exercising and sedentary animals is limited. Cognitive functions change during exercise and the aim of this study was to investigate rat protein levels of the protein machinery in the hippocampus, the main cognitive brain area for spatial learning and memory, in exercising rats. Protein fluctuations may reflect functional variation during exercise. Male Sprague–Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats undertaking voluntary exercise from 5 months to 23 months and the third undertaking involuntary exercise on a treadmill from 5 months to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification assigning spots to proteins and determination of coomassie-densities was carried out. Heterogeneous nuclear ribonucleoprotein K, one protein variant of heat shock cognate 71 kDa protein and BAG family molecular chaperone regulator 5 showed differential protein levels in the three groups when a p-value of <0.005 was considered as statistically significant thus respecting multiple testing. The biological meaning of changed protein levels in hippocampus under different conditions of exercise is not known but warrants further investigation.
Keywords: Exercise; Hippocampus; Rat; Proteomics; Protein machinery
Structure ofl-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii by Haruhiko Sakuraba; Kazunari Yoneda; Issaku Asai; Hideaki Tsuge; Nobuhiko Katunuma; Toshihisa Ohshima (pp. 563-571).
The crystal structure of the highly thermostablel-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 Å resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an α+β capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a β-strand that runs parallel to the C-terminal strand. This results in the formation of an additional β-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO.
Keywords: l; -Aspartate oxidase; Sulfolobus tokodaii; Archaea; Hyperthermophile; Crystal structure; Thermostability