Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Proteins and Proteomics (v.1794, #3)
Protein aggregation kinetics, mechanism, and curve-fitting: A review of the literature by Aimee M. Morris; Murielle A. Watzky ⁎; Richard G. Finke ⁎ (pp. 375-397).
Protein aggregation is an important phenomenon that alternatively is part of the normal functioning of nature or, central to this review, has negative consequences via its hypothesized central role in neurodegenerative diseases. A key to controlling protein aggregation is understanding the mechanism(s) of protein aggregation. Kinetic studies, data curve-fitting, and analysis are, in turn, keys to rigorous mechanistic studies. The main goal of this review is to analyze and report on the primary literature contributions to protein aggregation kinetics, mechanism, and curve-fitting. Following a brief introduction, the multiple different physical methods that have been employed to follow protein aggregation are presented and briefly discussed. Next, key information on the starting proteins and especially the products, and any detectable intermediates, involved in protein aggregation are presented. This is followed by tabulation (in the ) and discussion (in the main text), of the many approaches in the literature striving to determine the kinetics and mechanism of protein aggregation. It is found that these approaches can be broadly divided into three categories: (i) kinetic and thermodynamic, (ii) empirical, and (iii) other approaches. The first two approaches are the main focus of the present contribution, their goal being curve-fitting the available kinetic data and obtaining quantitative rate constants characterizing the nucleation, growth, and any other parts of the overall aggregation process. The large literature of protein aggregation is distilled down to five classes of postulated mechanisms: i) the subsequent monomer addition mechanism, ii) the reversible association mechanism, iii) prion aggregation mechanisms, iv) an “Ockham's razor”/minimalistic model first presented in 1997 and known as the Finke–Watzky 2-step model, and v) quantitative structure activity relationship models. These five classes of mechanisms are reviewed in detail in historical order; where possible corresponding kinetic equations, and fits to aggregation data via the proposed mechanisms, are analyzed and discussed. The five classes of mechanisms are then analyzed and discussed in terms of their similarities and differences to one another. Also included is a brief discussion of selected empirical approaches used to investigate protein aggregation. Three problem areas in the protein aggregation kinetic and mechanistic studies area are identified, and a section is provided en route to moving the field forward towards the still unachieved goal of unequivocal elucidation of the mechanism(s) of protein aggregation.
Keywords: Protein aggregation; Literature review; Kinetics; Mechanism; Curve-fitting
Transition of haemoglobin between two tertiary conformations: Inositol hexakisphosphate increases the transition constant and the affinity of sheep haemoglobin for 5,5′-dithiobis(2-nitrobenzoate) by Kehinde Onwochei Okonjo ⁎; Idowu Adeogun; J. Oyebamiji Babalola (pp. 398-409).
The equilibrium constant ( Kequ) for the reaction of 5,5′-dithiobis(2-nitrobenzoate) — DTNB — with the CysF9[93]β sulphydryl group of the haemoglobins of the sheep decreases by about two orders of magnitude between pH≈5.6 and 9.2: from a mean of 7.2±1 to a mean of 0.044±0.01. Calculations from the pH dependence of Kequ show that in ther⇌t tertiary conformational transition of haemoglobin thet isomer population is 50.7 and 61.8% for the major and minor haemoglobins, respectively. In the presence of inositol hexakisphosphate (inositol-P6), Kequ increases for both haemoglobins by about an order of magnitude through most of the pH range. Thet isomer population also increases to 82.1 and 79.6% for the major and minor haemoglobins, respectively. These results indicate that inositol-P6 increases the affinity of the sulphydryl for DTNB by increasing the population of thet isomer. It is highly probable that a minimum four-state model that includes ther⇌t transition is required for a full understanding of haemoglobin function.
Keywords: Haemoglobin tertiary conformation; Transition constant; Inositol hexakisphosphate; CysF9[93]β sulphydryl; 5,5′-dithiobis(2-nitrobenzoate) affinity
Role of protein surface charge in monellin sweetness by Wei-Feng Xue; Olga Szczepankiewicz; Eva Thulin; Sara Linse; Jannette Carey (pp. 410-420).
A small number of proteins have the unusual property of tasting intensely sweet. Despite many studies aimed at identifying their sweet taste determinants, the molecular basis of protein sweetness is not fully understood. Recent mutational studies of monellin have implicated positively charged residues in sweetness. In the present work, the effect of overall net charge was investigated using the complementary approach of negative charge alterations. Multiple substitutions of Asp/Asn and Glu/Gln residues radically altered the surface charge of single-chain monellin by removing six negative charges or adding four negative charges. Biophysical characterization using circular dichroism, fluorescence, and two-dimensional NMR demonstrates that the native fold of monellin is preserved in the variant proteins under physiological solution conditions although their stability toward chemical denaturation is altered. A human taste test was employed to determine the sweetness detection threshold of the variants. Removal of negative charges preserves monellin sweetness, whereas added negative charge has a large negative impact on sweetness. Meta-analysis of published charge variants of monellin and other sweet proteins reveals a general trend toward increasing sweetness with increasing positive net charge. Structural mapping of monellin variants identifies a hydrophobic surface predicted to face the receptor where introduced positive or negative charge reduces sweetness, and a polar surface where charges modulate long-range electrostatic complementarity.
Keywords: Abbreviations; Mn; monellin; scMn; single-chain monellin; WT; wildtype; pWT; pseudo-wildtype; CD; circular dichroism; NMR; nuclear magnetic resonance; HSQC; heteronuclear single-quantum correlation spectroscopyTaste receptor; Aftertaste; Electrostatic acceleration; Diffusion control; Brazzein; Thaumatin; Neoculin; Lysozyme
The modification of α-synuclein by dicarbonyl compounds inhibits its fibril-forming process by Daekyun Lee; Chang Wook Park; Seung R. Paik; Kwan Yong Choi ⁎ (pp. 421-430).
Oxidative modification of α-synuclein (αSyn) was reported to have significant effects on its amyloidogenic properties. Dicarbonyl compounds are metabolites accumulated by various oxidative processes in the intracellular environment. In this study, two dicarbonyl compounds, methylglyoxal (MGO) and glyoxal (GO), were investigated for their effects on the structural and fibril-forming properties of αSyn. Both compounds were found to induce the oligomerization of αSyn. By adding substoichiometric amounts of αSyn modified by MGO or GO, the fibrillization of αSyn was substantially inhibited. The heterogeneously-modified αSyns were separated into three fractions: monomers, oligomers, and high molecular mass oligomers. When each modified αSyn species was used to seed fibril formation, protein fibrillization was significantly suppressed. Temperature scanning and interactions with liposomes revealed that both MGO- and GO-modified monomers were not as susceptible as the unmodified αSyn to conformational changes into partially folded intermediates and α-helixes. Our observations suggest that dicarbonyl modification of αSyn reduces conformational flexibility of the protein, thereby contributing to a reduction in the ability of αSyn to form fibrils, and the modified protein inhibits the fibrillization of the unmodified αSyn.
Keywords: α-synuclein; Oxidative stress; Methylglyoxal; Glyoxal; AGE product; Fibrillization
Mass spectrometric demonstration of the presence of liver carnitine palmitoyltransferase-I (CPT-I) in heart mitochondria of adult rats by Anne M. Distler; Janos Kerner; Charles L. Hoppel (pp. 431-437).
The carnitine palmitoyltransferase-I (CPT-I) enzymes catalyze the regulated step in overall mitochondrial fatty acid oxidation. The liver and muscle isoforms are expressed in liver and skeletal muscle respectively with the isoforms exhibiting different kinetic properties and apparent molecular weight masses. In contrast, the heart expresses both isoforms at the mRNA level. However, for the expression of the liver isoform at the protein level only indirect evidence is available, such as tagging with radiolabeled CPT-I inhibitors followed by SDS-PAGE separation and kinetic analysis using inhibitors. The importance of fatty acid oxidation in the heart and the potential regulation via the liver isoform of CPT-I demands proof of the liver isoform in the heart. Using a proteomic approach in the present study we demonstrate that rat heart mitochondria (a) contain both the muscle and liver isoforms; (b) both proteins retain their C- and N-termini; (c) the N-terminal alanine residues are acetylated; (d) and in rat heart mitochondria the liver isoform is phosphorylated on tyrosine 281. By providing amino acid sequence information this is the first unequivocal demonstration that the liver isoform of CPT-I is expressed at the protein level in adult rat heart mitochondria and that the apparent smaller molecular size of the muscle isoform is not due to proteolytic truncation.
Keywords: Carnitine palmitoyltransferase-I; Liver and muscle isoform; Rat heart; Phosphorylation; Mitochondria; Proteomics; Mass spectrometry
Molecular determinants of substrate and inhibitor specificities of the Penicillium griseofulvum family 11 xylanases by Marine Cervera Tison; Gwénaëlle André-Leroux; Mickaël Lafond; Jacques Georis; Nathalie Juge; Jean-Guy Berrin (pp. 438-445).
Penicillium griseofulvum possesses two endo-(1,4)-β-xylanase genes, PgXynA and PgXynB, belonging to family 11 glycoside hydrolases. The enzymes share 69% identity, a similar hydrolysis profile i.e. the predominant production of xylobiose and xylotriose as end products from wheat arabinoxylan and a specificity region of six potential xylose subsites, but differ in terms of catalytic efficiency which can be explained by subtle structural differences in the positioning of xylohexaose in the PgXynB model. Site-directed mutagenesis of the “thumb” region revealed structural basis of PgXynB substrate and inhibitor specificities. We produced variants displaying increased catalytic efficiency towards wheat arabinoxylan and xylo-oligosaccharides and identified specific determinants in PgXynB “thumb” region responsible for resistance to the wheat xylanase inhibitor XIP-I. Based on kinetic analysis and homology modeling, we suggested that Pro130PgXynB, Lys131PgXynB and Lys132PgXynB hamper flexibility of the loop forming the “thumb” and interfere by steric hindrance with the inhibitor.
Keywords: Abbreviations; A/X; arabinose to xylose ratio; DNS; Dinitrosalicylic acid; GH; Glycoside hydrolase; GST; glutathione-; S; -transferase; HPAEC-PAD; High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection; LVAX; Low viscosity wheat arabinoxylan; MALDI-MS; Matrix-Assisted Laser Desorption Ionization/Mass Spectrometry; PfXynC; xylanase C from; Penicillium funiculosum; PgXynA; xylanase A from; Penicillium griseofulvum; PgXynB; xylanase B from; Penicillium griseofulvum; TAXI; Triticum aestivum; xylanase inhibitor; TLXI; thaumatin-like xylanase inhibitor; XIP-I; xylanase inhibitor protein I; X1; xylose; X2; xylobiose; X3; xylotriose; X4; xylotetraose; X5; xylopentose; X6; xylohexaoseGlycoside hydrolase family 11; Wheat arabinoxylan; Xylo-oligosaccharide; Site-directed mutagenesis; Xylanase inhibitor; Homology modeling
Proteomic analysis of human macrophages exposed to hypochlorite-oxidized low-density lipoprotein by Jeong Han Kang; Hyun Su Ryu; Hyun Tae Kim; Su Jin Lee; Ung-Kyu Choi; Yong Bok Park; Tae-Lin Huh; Myung-Sook Choi; Tae-Cheon Kang; Soo Young Choi; Oh-Shin Kwon ⁎ (pp. 446-458).
The invasion of monocytes through the endothelial wall of arteries and their transformation from macrophage into form cells has been implicated as a critical initiating event in atherogenesis. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) treatment, and can be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). To identify proteins potentially involved in atherosclerotic processes, we performed a proteomic analysis of THP-1 macrophages exposed to oxLDL generated by treatment with native LDL with hypochlorous acid/hypochlorite (HOCl/OCl−). We detected more than a thousand proteins, of which 104 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF) and the NCBI database. The largest differences in expression were observed for bifunctional purine biosynthesis protein, vacuolar protein sorting 33A, breast carcinoma amplified sequence, adenine phosphoribosyltransferase, and tropomyosin alpha 3 chain. Interestingly, many apoptotic proteins such as lamin B1, poly (ADP-ribose) polymerase, Bcl-2 related protein A1 and vimentin were identified by MALDI–TOF analysis. Identities were confirmed by matching the sequence of several tryptic peptides using MALDI–TOF/TOF MS, Western blot analyses and immunofluorescent microscopy. The data described here will contribute to establishing a functional profile of the human macrophage proteome. Furthermore, the proteins identified in this study are attractive candidates for further biomarkers involved in the pathogenesis of atherosclerosis.
Keywords: Abbreviations; PMA; phorbol 12-myristate 13-acetate; oxLDL; oxidized low-density lipoprotein; DAPI; 4′-6-Diamidino-2-phenylindole; DIC images; Differential Interference Contrast imageTHP-1 cell; Macrophage; Proteome; Oxidized low-density lipoprotein; Two-dimensional gel electrophoresis
Differential proteomic analysis of soluble extracellular proteins reveals the cysteine protease and cystatin involved in suspension-cultured cell proliferation in rice by Lihong Tian; Lei Zhang; Jie Zhang; Yun Song; Yi Guo ⁎ (pp. 459-467).
Extracellular matrix proteins play crucial roles in plant development, morphogenesis, cell division, and proliferation. To identify extracellular proteins that regulate cell growth, the soluble proteins of extracellular matrix were extracted from suspension-cultured rice cells for different lengths of time. The extract obtained from 3-d cultures was found to increase cells' fresh weight, while extracts from 6-d and 9-d cultures showed no effect on cells' growth. A comparative proteomic analysis was used to identify soluble extracellular proteins differentially expressed between 3 and 6 days in suspension-cultured cells. Ten unique protein spots were isolated and identified by mass spectrometry. Among them, included a cysteine protease (OsCP) and a putative cysteine protease inhibitor (cystatin, OC-I). OsCP has been down regulated in vivo using RNAi transgenic lines. The fresh and dry weight growth rates of OsCP RNAi cell lines were lower than empty vector control. Recombinant protein of OC-I could inhibit the OsCP protease activity, also it could inhibit the weight increase of suspension-culture cell as well as extracellular protease activity. These results suggest that OsCP and OC-I may be involved in the process of suspension-cultured rice cells proliferation.
Keywords: Abbreviations; CBB; Coomassie Brilliant Blue; CM; conditioned medium; 2-DE; two-dimensional electrophoresis; ECM; extracellular matrix; IEF; isoelectric focusing; PSK; phytosulfokine; PVDF; polyvinylidene difluorideCell proliferation; Cystatin; Cysteine protease; Soluble extracellular protein; Suspension-cultured rice cell
Putative “acylaminoacyl” peptidases from Streptomyces griseus and S. coelicolor display “aminopeptidase” activities with distinct substrate specificities and sensitivities to reducing reagent by Hirokazu Usuki; Yoshiko Uesugi; Masaki Iwabuchi; Tadashi Hatanaka (pp. 468-475).
Aminopeptidases from Streptomyces griseus (SGRAP) and S. coelicolor (SCOAP) were cloned and characterized to clarify their biochemical characteristics. Although both enzymes had been annotated as putative oligopeptidases of family S9 enzymes, they showed “aminopeptidase” activities, not “oligopeptidase” activities. Although their deduced amino acid sequences showed high similarity (69% overall sequence homology), they showed distinct substrate specificities and sensitivities to the reducing reagent dithiothreitol (DTT). The reaction pH and addition of DTT dramatically affected the substrate preference of SGRAP. Furthermore, SCOAP selectively hydrolyzed phenyalanine p-nitroanilide (Phe- pNA) in the presence or absence of DTT. The chimera protein between SGRAP and SCOAP was constructed to identify the region responsible for the properties described above. Furthermore, Cys409 of SCOAP was identified as a functional residue responsible for activation by reducing reagent DTT.
Keywords: Prolyl oligopeptidase; Phenylalanylaminopeptidase; Serine peptidase; Actinomycete; Substrate recognition property
Proteomic analysis of protein tyrosine nitration after ischemia reperfusion injury: Mitochondria as the major target by Bin Liu; Arun K. Tewari; Liwen Zhang; Kari B. Green-Church; Jay L. Zweier; Yeong-Renn Chen; Guanglong He (pp. 476-485).
Endothelial nitric oxide synthase-derived NO and its derivative, peroxynitrite (ONOO−), suppresses oxygen consumption by nitration of mitochondrial proteins after reperfusion. However, very few nitrated proteins are identified to date. In this paper, ischemia/reperfusion (I/R) injury was induced in mouse heart by ligation and release of the left anterior descending coronary artery. Western blotting showed that tyrosine nitration was higher in I/R hearts. Nitrated proteins were identified by capillary-liquid chromatography-nanospray tandem mass spectrometry. A total of 23 proteins were identified as being nitrated after I/R and 10 of them were from mitochondria. The nitrated mitochondrial proteins included 4 subunits from the oxidative phosphorylation system (the 24 and the 30 kDa subunits of complex I, the Rieske ISP of complex III, and the α subunit of ATP synthase), five enzymes in the matrix, and voltage-dependent anion channel. In purified complex I treated with ONOO−, 3-NT was identified locating at the residue of Y247 of the 30 kDa subunit and the residues of Y47, Y53 of the 49 kDa subunit. In conclusion, I/R induced protein nitration and mitochondrial proteins were the major targets. Selective nitration of proteins from the oxidative phosphorylation system at the beginning of reperfusion may contribute to the suppression of oxygen consumption.
Keywords: Peroxynitrite; Nitric oxide; Oxidative phosphorylation; Energy metabolism; Oxygen consumption
Heterooligomeric complexes formed by human small heat shock proteins HspB1 (Hsp27) and HspB6 (Hsp20) by Olesya V. Bukach; Alisa E. Glukhova; Alim S. Seit-Nebi; Nikolai B. Gusev ⁎ (pp. 486-495).
Formation of heterooligomeric complexes of human small heat shock proteins (sHsp) HspB6 (Hsp20) and HspB1 (Hsp27) was analyzed by means of native gel electrophoresis, analytical ultracentrifugation, chemical cross-linking and size-exclusion chromatography. HspB6 and HspB1 form at least two different complexes with apparent molecular masses 100–150 and 250–300 kDa, and formation of heterooligomeric complexes is temperature dependent. These complexes are highly mobile, easily exchange their subunits and are interconvertible. The stoichiometry of HspB1 and HspB6 in both complexes is close to 1/1 and smaller complexes are predominantly formed at low, whereas larger complexes are predominantly formed at high protein concentration. Formation of heterooligomeric complexes does not affect the chaperone-like activity of HspB1 and HspB6 if insulin or skeletal muscle F-actin was used as model protein substrates. After formation of heterooligomeric complexes the wild type HspB1 inhibits the rate of phosphorylation of HspB6 by cAMP-dependent protein kinase. The 3D mutant mimicking phosphorylation of HspB1 also forms heterooligomeric complexes with HspB6, but is ineffective in inhibition of HspB6 phosphorylation. Inside of heterooligomeric complexes HspB6 inhibits phosphorylation of HspB1 by MAPKAP2 kinase. Thus, in heterooligomeric complexes HspB6 and HspB1 mutually affect the structure of each other and formation of heterooligomeric complexes might influence diverse processes depending on small heat shock proteins.
Keywords: Abbreviations; DTT; dithiothreitol; 3D mutant of HspB1; triple S15,78,82D mutant of HspB1 mimicking phosphorylation by MAPKAP2 kinase; FRET; fluorescent resonance energy transfer; GST; glutathione-S-transferase; MBP; maltose-binding protein; PMSF; phenylmethane sulfonyl fluoride; sHsp; small heat shock protein(s)Small heat shock protein; Homo- and heterooligomeric complex; Phosphorylation; Chaperone-like activity
Redox properties and crystal structures of a Desulfovibrio vulgaris flavodoxin mutant in the monomeric and homodimeric forms by Andrea Fantuzzi; Roberto Artali; Gabriella Bombieri; Nicoletta Marchini; Fiorella Meneghetti; Gianfranco Gilardi; Sheila J. Sadeghi; Davide Cavazzini; Gian Luigi Rossi ⁎ (pp. 496-505).
The mutant S64C of the short-chain flavodoxin from Desulfovibrio vulgaris has been designed to introduce an accessible and reactive group on the protein surface. Crystals have been obtained of both the monomeric and homodimeric forms of the protein, with the cofactor FMN in either the oxidized or the one electron-reduced (semiquinone) state, and the structures have been determined to high resolution. The redox properties of the different species have been investigated and the variations observed with respect to wild type have been related to the structural changes induced by the mutation and S–S bridge formation.
Keywords: Desulfovibrio vulgaris; flavodoxin; Flavodoxin mutant; Homodimer; Crystal structure; Redox potential
Phospholipids influence the aggregation of recombinant ovine prions by Kirill Tsiroulnikov; Yuliya Shchutskaya; Vladimir Muronetz; Jean-Marc Chobert; Thomas Haertlé ⁎ (pp. 506-511).
The transformation of prion protein (PrP) into its insoluble amyloid form correlates with neurodegeneration in transmissible spongiform encephalopathies. PrP is connected to the neuronal membrane by a covalently-linked glycosylphosphatidylinositol (GPI) anchor. The current study demonstrates that phosphatidylinositol and phosphatidylethanolamine in low concentrations (0.5–50 μM) stimulate rapid unlimited aggregation of PrP. At a higher concentration (500 μM), lipid particles prevent the formation of large PrP aggregates and induce an increase in the β-sheet structure content of PrP protein. Thus, the liberation of PrP from the membrane and its direct interaction with its own GPI moiety, as well as with membrane lipids, can promote the formation of aggregated structures of PrP. The phospholipids studied are also able to upregulate the aggregation of oligomeric PrP forms (12-mers and 36-mers), the neurotoxicity of which has been reported recently. Low phosphatidylinositol concentrations induce these oligomers to form aggregates of smaller size when compared with aggregates formed directly from monomers. The inhibition of extensive aggregation observed at a high concentration of phosphatidylinositol (500 μM) results in both the formation of amyloids from PrP monomers and the interaction of protein molecules with lipid micelles. Thus, phospholipids are not only involved in the aggregation of prion monomers and their amyloidogenic conversion, but also regulate the aggregative status of prion oligomers already formed. Consequently, depending on their micellar status, phospholipids can either promote amyloidogenic conversion and conserve neurotoxic oligomeric forms (lipid micelles) or mediate the formation of large-size amorphous aggregates (non-micellar phospholipids).
Keywords: Abbreviations; PrP; prion protein; PrP; C; cellular isoform of PrP; PrP; Sc; scrapie isoform of PrP; PrP; recombinant ovine PrP; GPI; glycosylphosphatidylinositol; PI; phosphatidylinositol; MG; 1-oleoylglycerol; TG; 1,2,3-trioleoylglycerol; PA; phosphatidic acid; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phosphatidylserine; SM; sphingomyelin; CH; cholesterol; SA; stearic acid; OA; oleic acid; PK; proteinase K; SDS-PAGE; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DLS; dynamic light scattering; CD; circular dichroism; MOPS; 3-(N-morpholino) propane sulfonic acidPrion protein; Amyloid; Phosphatidylinositol
Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase by Anna Guðný Sigurðardóttir; Jóhanna Arnórsdóttir; Sigríður H. Thorbjarnardóttir; Guðmundur Eggertsson; Karsten Suhre; Magnús M. Kristjánsson ⁎ (pp. 512-518).
Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by ∼3 °C, both in T50% and Tm. Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity ( kcat) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DΔC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability ( Tm∼+1.5 °C) of the truncated enzyme (VPRΔC) as compared to the wild type VPR. Introduction of the N15D substitution into VPRΔC improved the thermal stability further by about 3 °C, or to about the same extent as in the wild type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties ( kcat) of the C-terminal truncated enzyme.
Keywords: Abbreviations; PMSF; phenylmethanesulfonyl fluoride; VPR; a subtilisin-like serine proteinase from a psychrotrophic; Vibrio; species; Suc-AAPF-NH-Np; Succinyl-AlaAlaProPhe-; p; -nitroanilide; MD; Molecular dynamicsIon pair; Stability; Kinetic property; Site directed mutagenesis; Cold adaptation; Subtilisin-like; Vibrio; -proteinase
Functional fusion mutant of Candida antarctica lipase B (CalB) expressed in Escherichia coli by Hyuk-Seong Seo; Seong-Eun Kim; Kyung-Yeon Han; Jin-Seung Park; Yong-Hwan Kim; Sang Jun Sim; Jeewon Lee ⁎ (pp. 519-525).
Candida antarctica lipase B (CalB) was functionally expressed in the cytoplasm of Escherichia coli Origami(DE3) with the N-terminus fusion of E. coli endogenous proteins. The previously-identified stress responsive proteins through comparative proteome analyses such as malate dehydrogenase (Mdh), spermidine/putrescine-binding periplasmic protein (PotD), and FKBP-type peptidyl–prolyl cis–trans isomerase (PPIases) (SlyD) dramatically increased the solubility of CalB in E. coli cytoplasm when used as N-terminus fusion partners. We demonstrated that Mdh, PotD, and SlyD were powerful solubility enhancers that presumably facilitated the protein folding of CalB. Moreover, among the various fusion mutants, Mdh-CalB showed the highest hydrolytic activity and was as biologically active as standard CalB. Similarly to the previous report, the electrophoretic properties of CalB indicate that CalB seems to form dimer-based oligomer structures. We evaluated the structural compatibility between the fusion partner protein and CalB, which seems to be of crucial importance upon the bioactive dimer formation of CalB and might affect the substrate accessibility to the enzyme active site, thereby determining the biological activities of the fusion mutants.
Keywords: CalB; Functional expression; Fusion mutant; Bioactivity; Escherichia coli
Assembly and function of AsGlu2 fibrillar multimer of oat β-glucosidase by Su-Nam Kwak; Sang-Yeob Kim; Sa-Ra Choi; In-Soo Kim ⁎ (pp. 526-531).
Oat β-glucosidase in plastid exists as a long fibrillar structure of AsGlu1 homomultimer (type I) and heteromultimer of AsGlu1 and AsGlu2 (type II). In spite of the high amino acid sequence homology of AsGlu1 and AsGlu2, AsGlu1 assembles into the fibrillar multimers but AsGlu2 forms a dimer when expressed in E. coli. A swapping analysis of AsGlu2 cDNA with AsGlu1 cDNA indicated that the C-terminal segment of AsGlu1 was critical for the fibrillar multimerization. A single substitution of glutamic acid-495 of AsGlu2 in the C-terminal region with lysine, an AsGlu1 counterpart amino acid for the glutamic acid-495, assembled the AsGlu2 into fibrillar homomultimers. The mutant AsGlu2 homomultimer was highly stable and had relatively faster electric mobility in native gel than the AsGlu1 homomultimer. Multimerization increased enzyme affinity to substrates.
Keywords: cDNA swapping; Enzyme structure; β-Glucosidase; Multimerization; Point mutation; Oat
Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of the pH induced changes in intrinsic fluorescence when iron is released by Nicholas G. James; Shaina L. Byrne; Anne B. Mason ⁎ (pp. 532-540).
Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome (∼pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to FeC hTF alone, the increase in the fluorescent signal from the preformed complex of FeC hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from FeC hTF. Quantum yield determinations of TFR, FeC hTF and the FeC hTF/TFR complex strongly support our interpretation of the kinetic data.
Keywords: Abbreviations; hTF; human serum transferrin; Fe; 2; hTF; diferric human serum transferrin; apo hTF; human serum transferrin lacking iron; Fe; C; hTF; recombinant monoferric hTF with iron in the C-lobe (Y95F/Y188F mutations preclude binding in the N-lobe), which has an N-terminal hexa His tag and is non-glycosylated; TFR; transferrin receptor; sTFR; soluble portion of the transferrin receptor expressed as a recombinant entity; 5-HTP; l; -5-hydroxytryptophan; DMEM-F12; Dulbecco's modified Eagle's medium-Ham F-12 nutrient mixture; BA; butyric acid; FBS; fetal bovine serum; UG; Ultroser G a serum substitute; GdHCl; guanidine HCl; BHK cells; baby hamster kidney cellsMetalloprotein; Protein–receptor interaction; Tryptophan analogue; Tryptophan fluorescence; Stopped-flow kinetic; BHK cell
Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and the anti-anti sigma factor RsfA by Shuja Shafi Malik; Amit Luthra; Ravishankar Ramachandran ⁎ (pp. 541-553).
M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and anti-anti sigma factors RsfA and RsfB to regulate the functions of the alternate sigma factor SigF. We have purified these proteins to characterize their molecular properties and interactions with UsfX. UsfX forms a stable complex with SigF that could be purified only after co-expressing the proteins in E.coli. Formation of the complex is nucleotide independent and apparently requires unknown in vivo factors. Fluorescence spectroscopy experiments suggest that the nucleotide binding sites of UsfX are distal to the protein-protein interaction interface. RsfA is a novel anti-anti sigma factor whose binding to UsfX is triggered by the reduction of an intrachain disulphide bond between Cys73–Cys109. The reduction is accompanied by an increase in the hydrodynamic radius of the protein. The UsfX–RsfA complex exhibits a novel stoichiometry of 2:1 compared to the 2:2 stoichiometry reported for other anti-anti-sigma factors. The role of the disulphide bond in complex formation was explored using molecular dynamics simulations. These studies support specific conformational changes that occur upon reduction of the Cys73–Cys109 bond of RsfA. This leads to a rearrangement that increases the interactions of a conserved His107 of UsfX with Cys109 of RsfA.
Keywords: Sigma factor F; RsfA; Anti-anti-sigma factor; M. tuberculosis; Protein–protein interaction; Redox potential
Structure and stability of a thioredoxin reductase from Sulfolobus solfataricus: A thermostable protein with two functions by Alessia Ruggiero; Mariorosario Masullo; Maria Rosaria Ruocco; Pasquale Grimaldi; Maria Angela Lanzotti; Paolo Arcari; Adriana Zagari; Luigi Vitagliano ⁎ (pp. 554-562).
Recent investigations have demonstrated that disulfide bridges may play a crucial role in the stabilization of proteins in hyperthermophilic organisms. A major role in the process of disulfide formation is played by ubiquitous proteins belonging to the thioredoxin superfamily, which includes thioredoxins (Trx), thioredoxin reductases (TrxR), and disulfide oxidases/isomerases (PDO/PDI). Here we report a characterization of the structure and stability of the TrxR ( SsTrxRB3) isolated from the archaeon Sulfolobus solfataricus. This protein is particularly interesting since it is able to process different substrates (Trxs and PDO) and it is endowed with an additional NADH oxidase activity. The crystal structure of the wild-type enzyme, of its complex with NADP and of the C147A mutant provides interesting clues on the enzyme function. In contrast to what is observed for class II TrxRs, in the structure of the oxidized enzyme, the FAD binding site is occupied by a partially disordered NAD molecule. In the active site of the C147A mutant, which exhibits a marginal NADH oxidase activity, the FAD is canonically bound to the enzyme. Molecular modeling indicates that a FAD molecule can be accommodated in the site of the reduced SsTrxRB3. Depending on the oxidation state, SsTrxRB3 can bind a different cofactor in its active site. This peculiar feature has been related to its dual activity. Denaturation experiments followed by circular dichroism indicate that electrostatic interactions play an important role in the stabilization of this thermostable protein. The analysis of the enzyme 3D-structure has also provided insights into the bases of SsTrxRB3 stability.
Keywords: Abbreviations; Trx; thioredoxin; TrxR; thioredoxin reductase; Ss; TrxRB3; S. solfataricus; TrxRB3; Ec; TrxR; E. coli; TrxR; At; TrxR; A. thaliana; TrxR; Hp; TrxR; H. pylori; TrxR; Mt; TrxR; M. tuberculosis; TrxR; FAD; flavin adenine dinucleotide; NADP; nicotinamide adenine dinucleotide phosphate; NAD; nicotinamide adenine dinucleotideOxidoreductase; Archaea; Protein function and stability; Cofactor binding; NADH oxidase
Oxidation of Cys278 of ADH I isozyme from Kluyveromyces lactis by naturally occurring disulfides causes its reversible inactivation by Tonino Bucciarelli; Michele Saliola; Fabrizia Brisdelli; Argante Bozzi; Claudio Falcone; Carmine Di Ilio; Filippo Martini (pp. 563-568).
The inactivation of the homotetrameric cytosolic alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) by naturally occurring disulfides, oxidized glutathione, cystine and cystamine, was studied. The inactivation was fully reversed by dithiothreitol. The nicotinamide coenzyme, but not the substrate ethanol, protected KlADH I from inactivation. Gel filtration experiments and SDS-PAGE analysis, also, revealed that enzyme inactivation coincides with inter-subunits disulfide bond formation which are noticeably enhanced after prolonged oxidation with GSSG. Moreover, oxidized KlADH I, as its reduced state, retained the tetrameric stucture and appears mainly as a dimer under non-reducing SDS-PAGE. Conversely, KlADH I Cys278Ile mutant is unaffected by disulfides treatment. Therefore, in vitro, KlADH I wild-type could exist in two reversible forms: reduced (active) and oxidized (inactive), in which the Cys278 residues of each tetramer are linked by disulfide bonds. The redox state of KlADH I could represent the path for modulating its activity and then a regulatory step of glycolysis under hypoxic conditions. It might be hypothesized that KlADH I could represent an important target in redox signaling of Kluyveromyces lactis cell by inhibiting, under oxidative stress, the glycolytic pathway in favor of the pentose-phosphate shunt to restore its reducing potential.
Keywords: Kluyveromyces lactis; alcohol dehydrogenase I; Enzyme inactivation; Inter-subunits disulfide bond; Enzyme oxidation
No effect of covalently linked poly(ethylene glycol) chains on protein internal dynamics by Margherita Gonnelli; Giovanni B. Strambini ⁎ (pp. 569-576).
Poly(ethylene glycol) or PEG is a hydrophilic polymer that covalently linked to therapeutical proteins may significantly increase their pharmacological properties. Despite the extensive production of PEG-conjugated proteins the effects of the polymer on the protein structure and dynamics is poorly understood, making the production of active biomaterials a largely unpredictable process. The present investigation examines the effects of 5 k and 20 k PEG on the internal flexibility of Ribonuclease T1, the mutant C112S of azurin from Pseudomonas aeruginosa, alcohol dehydrogenase and alkaline phosphatase, native and Zn-depleted. These systems encompass structural domains that range from rather superficial, flexible sites to deeply buried, rigid cores. The approach is based on three sensitive parameters related to the phosphorescence emission of internal Trp residues, namely, the intrinsic room-temperature phosphorescence lifetime ( τ0) that reports on the local flexibility of the protein matrix around the chromophore and the bimolecular rate constant ( kq) for the quenching of phosphorescence by O2 and by acrylamide in solution, which are related to the diffusion of these solutes through the protein fold. The results obtained by these three independent, intrinsic probes of protein structure-dynamics concur that mono-PEGylation does not detectably perturb the conformation and dynamics of the protein native fold, over a wide temperature range. The implication is that protein motions are essentially not coupled to the polymer and that adverse effects of chemical modification on biological function are presumably owed to steric hindrance by PEG units blocking the access to sites critical for molecular recognition.
Keywords: Abbreviations; Caz; mutant C112S of azurin from; Pseudomonas aeruginosa; NATA; N; -acetyl-tryptophanamide; LADH; horse liver alcohol dehydrogenase; AP; alkaline phosphatase; apoAP; Zn-depleted alkaline phosphatase; RNaseT; 1; Ribonuclease T1PEGylated protein; Protein dynamic; Phosphorescence; PEG