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BBA - Proteins and Proteomics (v.1814, #4)

Editorial Board (pp. i).

Solution structure of BTK-2, a novel hK_v1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus by G. Senthil Kumar; Sanjeev Upadhyay; M.K. Mathew; Siddhartha P. Sarma (pp. 459-469).

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK_v1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C^α, C′) and all heavy atoms are 0.81±0.23Å and 1.51±0.29Å respectively. The backbone dihedral angles (ϕ and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α–β–β structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure–function relationships among this important class of voltage-gated potassium channel inhibiting peptides.► Recombinantly derived biologically active three-disulfide scorpion toxin. ► Solution structure of the folded toxin determined using multidimensional NMR spectroscopy. ► Surface properties essential for Kv 1.1 channel activity.

Keywords: Abbreviations; BTK-2; scorpion toxin isolated from; Mesobuthus tamulus; NOESY-HSQC; nuclear Overhauser effect spectroscopy-heteronuclear single quantum coherence; RMSD; root-mean square deviation; HPLC; high performance liquid chromatographyNMR spectroscopy; Isotopically enriched peptide; Disulfide rich toxin; Solution structure; Folding of disulfide rich peptide

Proteomic identification of rhythmic proteins in rice seedlings by Heeyoun Hwang; Man-Ho Cho; Bum-Soo Hahn; Hyemin Lim; Yong-Kook Kwon; Tae-Ryong Hahn; Seong Hee Bhoo (pp. 470-479).

Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes.►It is the proteomic analysis of rhythmic proteins in an important crop plant, rice. ►Approximately 12% of resolved proteins in 2-DE gels are found to show diurnal and/or circadian rhythmicity. ►Discrepancies of rhythmic proteins between proteomic and transcriptomic data suggest that translational and/or post-translational modification is important in the rhythmic phenomena of proteins and proteomic approach is valuable to understand biological rhythms.

Keywords: Circadian; Diurnal; Proteomic; Rhythm; Rice; Comparative analysis

Efficient reduction of Cys110 thiyl radical by glutathione in human myoglobin by Satoshi Nagao; Osamu Asami; Hiroyuki Yasui; Shun Hirota (pp. 480-486).

Human myoglobin (hMb) possesses a cysteine (Cys) residue which is rare among mammalian Mbs. To investigate the effects of this unique Cys residue at the amino acid position 110 (Cys110) on hMb reactions, we studied the reactions of wild type (WT) methMb and its alanine mutant at Cys110 (C110A) with H₂O₂, particularly in the presence of reduced glutathione (GSH) which is well known as a reducing agent. The formation rates of the ferryloxo (Fe(IV)=O) species by H₂O₂ under air were about the same for WT and C110A methMbs, whereas the protein decomposed more in the case of WT than C110A hMb. With the addition of GSH, hMb consumed H₂O₂ faster and decomposition of the protein decreased, where the effects were more prominent in WT than C110A hMb. The radicals produced by the reaction with H₂O₂ decreased significantly due to the addition of 1mM GSH in the case of WT hMb, but not in the case of C110A hMb. These results show that GSH reduces H₂O₂-induced protein decomposition due to reduction of the C110-thiyl radical in WT hMb by electron transfer.► Wild type human myoglobin decomposes more than the C110A protein by H₂O₂ under air. ► The radical of human myoglobin decreases significantly with reduced glutathione. ► Reduced glutathione reduces H₂O₂-induced protein decomposition of human myoglobin.

Keywords: Abbreviations; Cys; cysteine; Mb; myoglobin; WT; wild type; GSH; reduced glutathione; GSSG; glutathione disulfide; DMPO; 5,5-dimethyl-1-pyrroline; N; -oxide; TEMPO; 2,2,6,6-tetramethylpiperidine-; N; -oxyl; MALDI-TOF; matrix-assisted laser desorption/ionization-time-of-flightHuman myoglobin; Hydrogen peroxide; Thiyl radical; Glutathione; Electron transfer

Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes by Xin-Miao Fu; Bao Ting Zhu (pp. 487-495).

Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.► Both PDI and PDIp can inactivate thermally-unfolded RNase that preserves native disulfide bonds. ► The N-terminal active site of PDIp plays a principal role in inactivating RNase. ► Stable disulfide-linked complexes are formed between PDI/PDIp and RNase. ► RNase can be released and reactivated from the PDI/PDIp–RNase complexes.

Keywords: Abbreviations; PDI; protein disulfide isomerase; PDIp; pancreas-specific PDI homolog; RNase; bovine pancreas RNase A; BPTI; bovine pancreas trypsin inhibitor; BSA; bovine serum albumin; DTT; dithiothreitol; GSH; glutathione; GSSG; oxidized glutathione; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid)Protein folding; PDI; PDIp; RNase; Disulfide bond; Isomerase

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus by Daniela Tsikou; Catalina Stedel; Evangelia D. Kouri; Michael K. Udvardi; Trevor L. Wang; Panagiotis Katinakis; Nikolaos E. Labrou; Emmanouil Flemetakis (pp. 496-504).

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO₂) metabolism.► Two full-length cDNAs coding for nodule-enhanced α-type CAs from L. japonicus. ► This is the first report on the kinetic characterization of plant α-type CAs. ► Transcripts of both genes are highly up-regulated during nodule development. ► Both transcripts are localized in the central, cortex and vascular tissues. ► Roles of these enzymes in the context of CO₂ metabolism in nodules are discussed.

Keywords: Abbreviations; CA; carbonic anhydrase; dpi; days post inoculation; ORFs; open reading frames; MES; 2-(N-morpholino)ethanesulfonic acid; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; SDS-PAGE; Sodium dodecyl sulfate polyacrylamide gel electrophoresis; qRT-PCR; quantitative real time polymerase chain reaction; TILLING; Targeting Induced Local Lesions in GenomesCarbonic anhydrase; Lotus japonicus; Nodule; Symbiotic nitrogen fixation

Ribosomal protein S18e as a putative molecular staple for the 18S rRNA 3′-major domain core by Aleksey A. Ilin; Alexey A. Malygin; Galina G. Karpova (pp. 505-512).

Ribosomal protein S18e is a structural constituent of the 40S ribosomal subunit. We obtained recombinant human ribosomal protein S18e and studied its structural and functional properties. With the use of CD spectroscopy we showed that the protein secondary structure is mainly helical and stable in the neutral pH range and at low urea concentrations. Applying multiple sequence alignment, we revealed that the protein structure has characteristics of the eukaryotic members of the ribosomal protein S13p family with additional extensions in the N-terminal and central parts that contain α-helices according to our prediction. S18e binds specifically and independently to an RNA transcript corresponding to the evolutionary core of the 3′-major domain of 18S rRNA. Hydroxyl radical footprinting showed that the binding site of S18e on the 18S rRNA is similar in general to the binding site of S13p on the 16S rRNA in the 30S ribosomal subunit, albeit the rRNA regions attributed to binding of the eukaryote-specific extensions of S18e were also detected. With magnesium ion concentration close to cellular conditions (2mM), protein binding caused substantial rearrangements in the rRNA transcript making it compact in such a manner that helices H29/H30 and H41–H43 form a bundle resembling their arrangement in the ribosome. Thus, S18e seems to act as a molecular staple fixing the 18S rRNA 3′-major domain core.► S18e binds specifically to 18S rRNA without assistance from other ribosomal proteins. ► S18e fixes the structure of the 18S rRNA 3′-major domain core acting as a staple. ► The eukaryote-specific extensions of S18e are involved in binding to the 18S rRNA. ► S18e is capable of folding the rRNA structure under a low Mg²⁺ concentration.

Keywords: Human ribosomal protein S18; Ribosomal protein S13p; 3′ Domain of 18S rRNA; Hydroxyl radical footprinting; 40S ribosomal subunit structure; 40S ribosomal subunit assembly

Structural and functional studies of the human selenium binding protein-1 and its involvement in hepatocellular carcinoma by Raffaele Raucci; Giovanni Colonna; Eliana Guerriero; Francesca Capone; Marina Accardo; Giuseppe Castello; Susan Costantini (pp. 513-522).

The reduced expression of human selenium binding protein-1 (SELENBP1) has been reported for some human cancers. In this work we have estimated a reduced SELENBP1 expression by immunohistochemistry for the first time also in liver tissues of patients with hepatocarcinoma (HCC). Since the structure–function relationships of SELENBP1 are unknown, we have performed computational and experimental studies to have insight on the structural features of this protein focusing our attention on the properties of cysteines to assess their ability to interact with selenium. We have performed CD studies on the purified protein, modeled its three-dimensional structure, studied the energetic stability of the protein by molecular dynamics simulations, and titrated the cysteines by DTNB (5,5′-dithiobis (2-nitrobenzoic acid). The secondary structure content evaluated by CD has been found similar to that of 3D model. Our studies demonstrate that (i) SELENBP1 is an alpha-beta protein with some loop regions characterized by the presence of intrinsically unordered segments, (ii) only one cysteine (Cys57) is enough exposed to solvent, located on a loop and surrounded by charged and hydrophobic residues, and can be the cysteine able to bind the selenium. Furthermore, during the molecular dynamics simulation at neutral pH the loop containing Cys57 opens and exposes this residue to solvent, confirming that it is the best candidate to bind the selenium. Experimentally we found that only one cysteine is titratable by DTNB. This supports the hypothesis that Cys57 is a residue functionally important and this may open new pharmacological perspectives.►SELENBP1 belongs to “selenium binding proteins” family. ►Selenium is an important oligoelement. ►Expression of SELENBP1 in liver tissues of patients with hepatocarcinoma is reduced. ►SELENBP1 is an alpha-beta protein with some intrinsically unordered loop regions. ►Cys57 is the only exposed cysteine with high probability of binding to selenium.

Keywords: Selenium binding protein; Structural model; Molecular dynamics; Hepatocarcinoma; Hepatocarcinoma prognosis

Corrigendum to “Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli” [Biochem. Biophys. Acta 1794 (2009) 1566–1572] by MeiZhi Weng; ZhongLiang Zheng; Wei Bao; YongJun Cai; Yan Yin; GuoLin Zou (pp. 523-523).

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BBA - Proteins and Proteomics (v.1814, #4)