Protein and Peptide Letters - Volume 14, Issue 4, 2007

The interaction of Cu(II) ion with small peptides has been an interesting subject to clarify the role of copper in detail. As various Cu(II)-oligopeptide complexes can also be good models for the active centers of metalloenzymes, complexes of tripeptide and tetrapeptides are frequently investigated instead of the complexes of large peptides. The histidine side-chains of various metalloproteins frequently take part in the copper(II) coordination. Accordingly, we studied the coordination of Cu(II) to the N and C terminal protected tripeptide ligands LA (Ac-HisGlyHis-NHMe), LB (Ac-HisAlaHis- NHMe) and LC (Ac-HisAibHis-NHMe) in aqueous solution potentiometrially in order to determine the effect of Cα methyl groups at middle residue acid on the ligation of the backbone NH and also on histidine's Nim of coordination. Species distribution curves indicates that in acidic pH, all three peptides behave as bidentate ligands and a macrochelate forms on the metal coordination with the two histidine imidazolyl N. This coordination remains unaffected with the +I effect of increasing CH3 groups at Cα of middle residue. In the pH range 4-8, the tridentate coordination from the peptide is seen in ligand LA and LB while it is absent in LC due to +I effect of two Cα methyl groups at middle residue as they makes Nterminal NH deprotonation difficult in this pH range and it takes place along with C terminal NH and only 4N coordinated species formed at higher pH. These 4N (Nim, N-, N-, Nim) coordinated species are formed by all the three ligands at higher pH values.

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense- related genes.To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 μM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade.Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 μM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 μM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.

We tested the ability of Taq DNA polymerase (Taq) to amplify long DNA fragments and showed that, if the conditions were set properly, Taq could successfully perform the “long PCR” up to 24 kb. The conditions include: (1) longer primers, (2) a 2-step cycling, and (3) a “long buffer.” We propose that the most important requirements are the survival rate of Taq at high temperatures and that of the primers against the 5' to 3' exonuclease activity of Taq.

Blomia tropicalis allergens are the most important mite allergens in tropical regions. Most of them only have 30-40% sequence identity with their Dermatophagoides counterparts and they share low IgE cross reactivity and exhibit different immunobiology. Unlike the pyroglyphid counterparts, Blo t 5 is the major allergen whereas Blo t 1 only has modest allergenicity.

Although the tertiary structures of mitochondrial cytochromes c (cyts c) seem to be remarkably similar, there are variations in their amino acid sequences, stability and functional properties. GdnHCl-induced unfolding experiments on engineered yeast and horse cyt c were carried out with the aim to to clarify, at molecular level, some aspects concerning the stability of this class of proteins. The results obtained are discussed in the light of the three-dimensional structures of the two proteins.

This study indicated that the N-terminus was dispensable for FtsY GTPase activity, and that the N-domain plays an essential role in the GTPase activity of the NG domain. In addition, the S.scoelicolor FtsY was able to restore function in an E. coli mutant. However, its NG domain was unable to play any roles.

We show that α-synuclein could assist the molecular activity of a ketoprofen-(R/S) nonspecific esterase from Archaeglobus Fulgidus. Specifically, several synthetic peptides from α-synuclein, each having random coil conformation in far-UV spectra, could protect the enzyme activity against stress conditions such as heat and organic solvents.

Conformational properties of two potentially β-turn forming peptides were determined using a strategy which combines MD simulations, IR and VCD spectroscopy and quantum chemical calculations. This strategy could be a useful alternative for solution conformational analysis of short flexible peptides and could help to identify VCD features which are as yet unknown.

Background: The molecular regulation of MAPKs and apoptosis was investigated in a model of hypoxiatolerance. Survival of neurons in Chrysemys picta bellii, an anoxia-tolerant turtle, involves a reduction in energy metabolism. The biochemical/physiological mechanisms of anoxia tolerance have been examined at the level of ion transport and ATP turnover. However, changes in the phosphorylation state of key enzymes and kinases, mainly, MAPKs, may occur during anoxia, thereby reversible protein phosphorylation could be a critical factor and major mechanism of metabolic reorganization for enduring anaerobiosis. Methods: If a turtle were to undergo hypoxia akin to that experienced in its native habitat, it was placed in a glass aquarium filled with water to within a half inch of the top. After the turtle was anesthetized, through extended hypoxia or anesthesia, the animal was sacrificed by decapitation. The brain was then excised and placed in anoxic artificial cerebrospinal fluid. Total protein extraction was performed by homogenizing brain in a buffer, followed by threonine and tyrosine phosphorylation determination of MAPKs, and caspase activity. Results: MAPKp38 was decreased after reoxygenation following 1 day and 1 week hypoxia. The effect of hypoxia on the phosphorylation of MAPKERK was biphasic: Enhancement at 5h and inhibition at 6 weeks. Pro-caspases 8/9 were unchanged by hypoxia until increasing at 6 weeks. Both pro-caspases were upregulated by reoxygenation at 1 day or 6 weeks hypoxia. Neither hypoxia nor reoxygenation induced the cleavage of pro-caspases 8/9 into p20 and p10, respectively. Furthermore, hypoxia induced Bax at 3 days and 1 week, and reoxygenation increased Bax ≈ 4-fold at 1 day. Although the expression of Bcl-2 was slightly increased by hypoxia, [Bcl-2] was 3-4-fold smaller in comparison with Bax. Conclusion: These results indicate that hypoxia up-regulates MAPKERK but not MAPKp38; hypoxia/reperfusion increases the expression of caspases and pro-apoptotic cofactors. The patterns of MAPK regulation suggest the significance of these kinases in cellular adaptation to oxygen deprivation with biomedical correlations, and thereby identify novel natural responsive signaling cofactors in Chrysemys picta bellii with potential pharmacologic and clinical applications.

Background: Pursuant to establishing the proteomic distribution of MAPKERK/MAPKp38 in the brain in a model of hypoxia-tolerance [Haddad, Protein Pept Lett, In press, 2007], I therein exclusively report the differential expression of MAPKJNK and related upstream and downstream kinases in various organs of the anoxia-tolerant turtle. Despite the fact that the aforementioned mechanisms involved dual expression of MAPKERK, the mechanistic distribution of MAPKJNK has not been previously unraveled. Changes in the phosphorylation state of MAPKs may occur during anoxia, thereby reversible protein phosphorylation could be a critical factor and major mechanism of metabolic reorganization for enduring anaerobiosis. Methods: If a turtle were to undergo hypoxia akin to that experienced in its native habitat, it was placed in a glass aquarium filled with water to within a half inch of the top. After the turtle was anesthetized, through extended hypoxia or anesthesia, the animal was sacrificed by decapitation. The brain and other organs were then excised and placed in anoxic artificial cerebrospinal fluid. Total protein extraction was performed by homogenizing various organs in a suitable buffer, followed by determination of the phosphorylation states of SEK-1/MKK-4, SAPK/MAPKJNK and c-Jun activating protein (AP)-1. Results: SEK-1/MKK-4 expression was mild in the cortex as compared with the manifold hypoxic (2h) induction in the liver. Continuous imposition of hypoxia (1 day - 1 week) increased the expression of SEK-1/MKK-4, thereafter declined at 3 weeks hypoxia. Hypoxia/reoxygenation weakly induced SEK-1/MKK-4 expression in cortex, in contrast with a strong induction in the liver, but not in other organs. Hypoxia (2h - 3 weeks) did not induce SAPK/MAPKJNK expression in cortex, despite prominent increase in liver, with mild reoxygenation effect. The normoxic induction of c-Jun AP-1 in cortex and rest of brain (ROB) was reduced with imposition of hypoxia (2h - 1 week). Furthermore, hypoxia (2h - 3 weeks) upregulated expression of c-Jun AP-1 in liver, heart and spleen, an effect abrogated with hypoxia/reoxygenation. Conclusion: These results indicate that hypoxia differentially up-regulates the expression of MAPKJNK-related cofactors with organ-specific distribution. Since these modules are involved with neuroprotection in Chrysemys picta bellii, the expression of MAPKs bears relative mechanisms of specific responses to hypoxia tolerance.

Di/oligomerization of G-protein coupled receptors (GPCRs) is well established, however very little is known regarding the interaction details. Current paper presents results of molecular dynamics simulations of theoretical model of rhodopsin tetramer with transducine (Gt) in lipid bilayer. Ligand-protein and receptor-receptor interactions have been proposed.

Improperly folded metal cofactor-containing proteins (e.g., copper chaperone for superoxide dismutase, CCS) are believed to play a key role in several protein-misfolding diseases (e.g., Alzheimer's disease or Amyotrophic Lateral Sclerosis) because under regular physiological conditions, metallochaperones activate or stabilize the native conformation of important metalloproteins (e.g., superoxide dismutase) in certain cellular processes. For an improved diagnosis and therapy of neurodegenerative diseases, new methodologies have to be developed that enable a well-defined differentiation between properly folded and inactive metalloproteins in clinical samples. In the literature it is reported that different high molecular mass metal-containing proteins were isolated in brain samples from Alzheimer's patients and in vegetables by using a 2-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. In the present article, selected results of these studies are scrutinized and compared with some results obtained by a standardized method termed ‘quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE)’. Conclusively, QPNC-PAGE is a highly efficient approach used by biochemists to resolve native and denatured metalloproteins (MW 6 - ≥ 200 kDa) in complex protein mixtures.

Snake venoms hydrolyze several phosphorylated substrates. However, not is clearly understood which enzyme( s) is (are) involved in these process. Here, we propose the existence of an independent ADPase activity. In addition, we studied the reactions mechanism of nucleotide hydrolysis. This system resembles membrane ecto-nucleotidases and acts with a multi enzymatic complex transforming ATP in adenosine without the accumulation of intermediates.

A new member of archaeal DNA polymerase from Pyrococcus furiosus was crystallized. Diffraction data to 3.1 Å of the selenomethionine-derivatized crystal were collected, and preliminary crystallographic study has been completed. The crystal belongs to the space group C2 with unit cell parameters of a = 93.2 Å, b = 124.9 Å, c = 87.7 Å, α = 90 , β = 109.7® , and γ = 90 . Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystal is estimated to be 54%, corresponding to a Matthews coefficient VM of 2.7Å3 Da-1.

PD-L1 is a highly glycosylated type 1 ribosome inactivating protein, from Phytolacca dioica leaves, with the peculiarity to act also as a DNase. PD-L1 has been successfully crystallized using vapour diffusion and seeding techniques. Crystals belong to the monoclinic C2 space group, with unit cell dimensions a=161.01, b=34.73, c=120.63 Å, β=127.99 . Two molecules are present in the asymmetric unit. Phase determination has been achieved using molecular replacement.