The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle
Sucharitakul, J. ; Tongsook, C. ; Pakotiprapha, D. ; Berkel, W.J.H. van; Chaiyen, P. - \ 2013
Journal of Biological Chemistry 288 (2013)49. - ISSN 0021-9258 - p. 35210 - 35221.
para-hydroxybenzoate hydroxylase - p-hydroxyphenylacetate 3-hydroxylase - 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase - steady-state - ornithine hydroxylase - vibrio-campbellii - crystal-structure - gentisic acid - in-vitro - mechanism
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10(6) m(-1) s(-1) (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s(-1). This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s(-1). 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s(-1). Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s(-1)) and hydroxylation (36 ± 2 s(-1)) partially control the overall turnover
FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase
Tischler, D. ; Schlömann, M. ; Berkel, W.J.H. van; Gassner, G.T. - \ 2013
FEBS Letters 587 (2013)23. - ISSN 0014-5793 - p. 3848 - 3852.
rhodococcus-opacus 1cp - phenol hydroxylase - wild-type - mechanism
StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.
Increased plasma citrulline in mice marks diet-induced obesity and may predict the development of the metabolic syndrome
Sailer, M. ; Dahlhoff, C. ; Giesbertz, P. ; Eidens, M.K. ; Wit, N.J.W. de; Rubio-Aliaga, I. ; Boekschoten, M.V. ; Müller, M.R. ; Daniel, H. - \ 2013
PLoS ONE 8 (2013)5. - ISSN 1932-6203
amino-acid transporter - skeletal-muscle cells - arginine bioavailability ratios - high-fat diet - insulin-resistance - l-alanine - protein - liver - secretion - mechanism
Article About the Authors Metrics Comments Related Content Abstract Introduction Results Discussion Materials and Methods Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “
Analysis of steady-state Förster resonance energy transfer data by avoiding pitfalls: Interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides.
Niranjan, Y. ; Ungureanu, D. ; Hammarén, H. ; Sanz-Sanz, A. ; Westphal, A.H. ; Borst, J.W. ; Silvennoinen, O. ; Hilhorst, M.H. - \ 2013
Analytical Biochemistry 442 (2013)2. - ISSN 0003-2697 - p. 213 - 222.
pseudokinase domain - protein-kinase - fluorescence - atp - binding - receptor - analogs - site - autophosphorylation - mechanism
Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2'/3'-(N-methyl-anthraniloyl)-adenosine-5'-triphosphate (MANT–ATP) and enzymes is widely used to determine affinities for ATP–protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT–ATP, MANT–ADP [2'/3'-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT–AMP [2'/3'-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT–ATP tightly with a Kd of 15 to 25 nM and excluded the presence of a second binding site. The affinity for MANT–ADP is also tight with a Kd of 50 to 80 nM, whereas MANT–AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT–ATP-¿-S [2'/3'-O-(N-methylanthraniloyl) adenosine-5'-(thio)- triphosphate] yielded a Kd of 30 to 50 nM. The methods demonstrated here are applicable to other enzyme–fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins.
Conformational landscapes of DNA polymerase I and mutator derivates establish fidelity checkpoints for nucleotide insertion
Hohlbein, J.C. ; Aigrain, L. ; Craggs, T.D. ; Bermek, O. ; Potapova, O. ; Shoolizadeh, P. ; Grindley, N.D.F. ; Joyce, C.M. ; Kapanidis, A.N. - \ 2013
Nature Communications 4 (2013). - ISSN 2041-1723 - 11 p.
single-molecule fret - resonance energy-transfer - probability-distribution analysis - alternating-laser excitation - klenow fragment - escherichia-coli - active-site - photon distribution - mechanism - dynamics
The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
Ultrasound-assisted MnO2 catalyzed homolysis of peracetic acid for phenol degradation: The assessment of process chemistry and kinetics
Rokhina, E.V. ; Makarova, K. ; Lathinen, M. ; Golovina, E.A. ; As, H. van; Virkutyte, J. - \ 2013
Chemical Engineering Journal 221 (2013). - ISSN 1385-8947 - p. 476 - 486.
wet peroxide oxidation - aqueous-solutions - free-radicals - mechanism - systems - water - decomposition - sonochemistry - intermediate - destruction
The combination of peracetic acid (PAA) and heterogeneous catalyst (MnO2) was used for the degradation of phenol in an aqueous solution in the presence of ultrasound irradiation (US). As a relevant source of free radicals (e.g. OH), peracetic acid was comprehensively studied by means of electron spin resonance (ESR) spin trapping (ST) techniques with the subsequent identification of free radicals by simulation based fitting (SBF) technique. The radical reaction mechanism, where hydroxyl radical was a primary product of OO bond rupture of PAA, was established taking into account radical reactions, occurring during sonolysis. The potential barriers and the reaction heat were determined by basic density function theory (DFT) calculations to estimate whether the proposed radical pathway is possible. The assessment and optimization of the process parameters for MnO2/PAA/US system to eliminate phenol was accomplished with experimental design. Fractional factorial design (FFD) was executed to relate the removal efficiency of phenol with process parameters such as catalyst and PAA concentrations, the presence of ultrasound and the reaction time. The comparative kinetic study of silent and ultrasound-assisted processes revealed the significant difference between these two processes that was mainly attributed to the complex radical system formed during PAA homolysis
Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation
Lindeboom, J.J. ; Lioutas, A. ; Deinum, E.E. ; Tindemans, S. ; Ehrhardt, D.W. ; Emons, A.M.C. ; Mulder, B. - \ 2013
Plant Physiology 161 (2013)3. - ISSN 0032-0889 - p. 1189 - 1201.
plant-cells - nitella-tasmanica - self-organization - gamma-tubulin - arabidopsis - nucleation - mechanism - orientation - dynamics - reveals
The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-¿-tubulin complex protein2-tagged ¿-nucleation complexes (¿-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving ¿-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.
A physical cross-linking process of cellulose nanofibril gels with shear-controlled fibril orientation
Fall, A.B. ; Lindstrom, S.B. ; Sprakel, J.H.B. ; Wagberg, L. - \ 2013
Soft Matter 9 (2013)6. - ISSN 1744-683X - p. 1852 - 1863.
dynamic light-scattering - microfibrillated cellulose - nanocomposites - polyelectrolyte - mechanism - networks - modulus
Cellulose nanofibrils constitute the smallest fibrous components of wood, with a width of approximately 4 nm and a length in the micrometer range. They consist of aligned linear cellulose chains with crystallinity exceeding 60%, rendering stiff, high-aspect-ratio rods. These properties are advantageous in the reinforcement components of composites. Cross-linked networks of fibrils can be used as templates into which a polymer enters. In the semi-concentrated regime (i.e. slightly above the overlap concentration), carboxy methylated fibrils dispersed in water have been physically cross-linked to form a volume-spanning network (a gel) by reducing the pH or adding salt, which diminishes the electrostatic repulsion between fibrils. By applying shear during or after this gelation process, we can orient the fibrils in a preferred direction within the gel, for the purpose of fully utilizing the high stiffness and strength of the fibrils as reinforcement components. Using these gels as templates enables precise control of the spatial distribution and orientation of the dispersed phase of the composites, optimizing the potentially very large reinforcement capacity of the nanofibrils.
The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A
Kuijk, S.J.A. van; Noll, K.S. ; Chikindas, M.L. - \ 2012
Letters in Applied Microbiology 54 (2012)1. - ISSN 0266-8254 - p. 52 - 58.
bacillus-subtilis - bacteriocins - antibiotics - mechanism - pathogen - growth - acid - ph
Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amylliquefaciens. The minimal inhibitory concentration of subtilosin against L. moilocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (Ali) and pH gradient (ApH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogencs growth at a concentration of 19 fig Subtilosin caused a partial depletion of the AT and had a similar minor effect on the ApIL There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogencs Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogcues Scott A differs from the one previously described for another human path()gen, Cam dnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species specific mode of action
Long- and Medium-Chain Fatty Acids Induce Insulin Resistance to a Similar Extent in Humans Despite Marked Differences in Muscle Fat Accumulation
Hoeks, J. ; Mensink, M.R. ; Hesselink, M.K.C. ; Ekroos, K. ; Schrauwen, P. - \ 2012
Journal of Clinical Endocrinology and Metabolism 97 (2012)1. - ISSN 0021-972X - p. 208 - 216.
human skeletal-muscle - intramyocellular lipid-content - prolonged exercise - ceramide content - obese subjects - oxidation - mechanism - men - diacylglycerol - sensitivity
Context: Animal studies revealed that medium-chain fatty acids (MCFA), due to their metabolic characteristics, are not stored in skeletal muscle and may therefore not give rise to potentially hazardous lipid species impeding insulin signaling. Objective: We here hypothesized that infusion of medium-chain triacylglycerols (MCT) in healthy lean subjects does not lead to ectopic fat accumulation and hence does not result in lipid-induced insulin resistance. Design and Methods: Nine healthy lean male subjects underwent a 6-h hyperinsulinemic-euglycemic clamp with simultaneous infusion of 1) a 100% long-chain triacylglycerols (LCT) emulsion, 2) a 50/50% MCT/LCT emulsion, or 3) glycerol in a randomized crossover design. Muscle biopsies were taken before and after each clamp. Results: MCT/LCT infusion raised plasma free fatty acid levels to a similar level compared with LCT infusion alone. Despite elevated free fatty acid levels, intramyocellular triacylglycerol (IMTG) levels were not affected by the MCT/LCT emulsion, whereas LCT infusion resulted in an approximately 1.6-fold increase in IMTG. These differences in muscle fat accumulation did not result in significant differences in lipid-induced insulin resistance between LCT (- 28%, P = 0.003) andMCT/LCT (-20%, P <0.001). Total skeletal muscle ceramide content as well as lactosyl-and glucosylceramide levels were not affected by any of the interventions. In addition, the distribution pattern of all ceramide species remained unaltered. Conclusions: Although we confirm that MCFA do not lead to ceramide and IMTG accumulation in skeletal muscle tissue in humans, they do induce insulin resistance. These results indicate that, in humans, MCFA may not be beneficial in preventing peripheral insulin resistance. (J Clin Endocrinol Metab 97: 208-216, 2012)
Campylobacter jejuni is highly susceptible to killing by chicken host defense peptide cathelicidin-2 and suppresses intestinal cathelicidin-2 expression in young broilers
Dijk, A. van; Herrebout, M. ; Tersteeg-Zijderveld, M.H.G. ; Tjeerdsma-van Bokhoven, J.L.M. ; Bleumink-Pluym, N. ; Jansman, A.J.M. ; Veldhuizen, E.J.A. ; Haagsman, H.P. - \ 2012
Veterinary Microbiology 160 (2012)3-4. - ISSN 0378-1135 - p. 347 - 354.
day-of-hatch - enteric infections - resistance - identification - colonization - pathogenesis - heterophils - mechanism - virulence - ll-37
Little is known about the interactions of chicken host defense peptides (HDPs) with Campylobacter jejuni in young chicks. To examine the role of the chicken HDP, cathelicidin-2 (CATH-2) in host-pathogen interactions we challenged 4-day-old Ross 308 broilers with a chicken-derived C jejuni isolate (WS356) and used the chicken pathogen Salmonella enterica Enteritidis phage type 4 (FGT1) as a reference. Immunohistochemical staining was used to localize CATH-2, C jejuni and Salmonella enteritidis. Intestinal CATH-2 mRNA expression levels were determined by quantitative PCR. Antibacterial activities of CATH-2 peptide against C. jejuni and S. enteritidis isolates were assessed in colony count assays. In contrast to S. enteritidis, C jejuni was not seen to attach to intestinal epithelium and C jejuni challenge did not result in recruitment of CATH-2 containing heterophils to the small intestinal lamina propria. Minimal inhibitory concentrations found for CATH-2 peptide against human- and chicken-derived C. jejuni isolates were similar (0.6-2.5 mu M) and much lower than for S. enteritidis (20 mu M). Compared to wild-type C. jejuni 81116, the lipooligosaccharide (LOS)-deficient 81116 Delta waaF mutant was much more susceptible to CATH-2. Interestingly, CATH-2 mRNA expression levels in the small intestine were significantly lower 48 h p.i. in C jejuni-challenged chicks. These findings indicate that human clinical and chicken-derived C jejuni are equally highly susceptible to chicken CATH-2 peptide and that C jejuni uses LOS to protect itself to some extent against HDPs. Moreover, suppression of intestinal CATH-2 expression levels may be part of the C. jejuni immune evasion strategy.
Influence of cell-to-cell variability on spatial pattern formation
Greese, B. ; Wester, K. ; Bensch, R. ; Ronneberger, O. ; Timmer, J. ; Huulskamp, M. ; Fleck, C. - \ 2012
IET Systems Biology 6 (2012)4. - ISSN 1751-8849 - p. 143 - 153.
stochastic gene-expression - biochemical reactions - lateral inhibition - single-cell - arabidopsis - noise - mechanism - differentiation - stability - tessellations
Many spatial patterns in biology arise through differentiation of selected cells within a tissue, which is regulated by a genetic network. This is specified by its structure, parameterisation and the noise on its components and reactions. The latter, in particular, is not well examined because it is rather difficult to trace. The authors use suitable local mathematical measures based on the Voronoi diagram of experimentally determined positions of epidermal plant hairs (trichomes) to examine the variability or noise in pattern formation. Although trichome initiation is a highly regulated process, the authors show that the experimentally observed trichome pattern is substantially disturbed by cell-to-cell variations. Using computer simulations, they find that the rates concerning the availability of the protein complex that triggers trichome formation plays a significant role in noise-induced variations of the pattern. The focus on the effects of cell noise yields further insights into pattern formation of trichomes. The authors expect that similar strategies can contribute to the understanding of other differentiation processes by elucidating the role of naturally occurring fluctuations in the concentration of cellular components or their properties
Cascade-mediated binding and bending of negatively supercoiled DNA
Westra, E.R. ; Nilges, B. ; Erp, P.B. ; Oost, J. van der; Dame, R.T. ; Brouns, S.J.J. - \ 2012
RNA Biology 9 (2012)9. - ISSN 1547-6286 - p. 1134 - 1138.
crispr-cas systems - immune-system - structural basis - rna - bacteria - archaea - defense - interference - recognition - mechanism
Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop
Picosecond Kinetics of Light Harvesting and Photoprotective Quenching in Wild-Type and Mutant Phycobilisomes Isolated from the Cyanobacterium Synechocystis PCC 6803
Tian, L. ; Gwizdala, M. ; Stokkum, I.H.M. van; Koehorst, R.B.M. ; Kirilovsky, D. ; Amerongen, H. van - \ 2012
Biophysical Journal 102 (2012)7. - ISSN 0006-3495 - p. 1692 - 1700.
orange carotenoid protein - chlorophyll-binding protein - energy-dissipation - photosystem-ii - molecular architecture - higher-plants - fluorescence - mechanism - organization - photoinhibition
In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCPr), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APCQ660) with a molecular quenching rate that is faster than (1 ps)-1. Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APCQ660 and OCP or by excitation energy transfer from APCQ660 to the S1 state of the carotenoid—a distinction that is very hard, if not impossible, to make in vivo.
Interdecadal North-Atlantic meridional overturning circulation variability in EC-EARTH
Wouters, B. ; Drijfhout, D. ; Hazeleger, W. - \ 2012
Climate Dynamics 39 (2012)11. - ISSN 0930-7575 - p. 2695 - 2712.
multidecadal climate variability - sea-surface temperature - ocean-atmosphere gcm - thermohaline circulation - gulf-stream - decadal variability - oscillation - model - mechanism - transport
The Atlantic meridional overturning circulation (AMOC) in a 600 years pre-industrial run of the newly developed EC-EARTH model features marked interdecadal variability with a dominant time-scale of 50–60 years. An oscillation of approximately 2 Sverdrup (1 Sv = 106 m3 s-1) is identified, which manifests itself as a monopole causing the overturning to simultaneously strengthen (/weaken) and deepen (/shallow) as a whole. Eight years before the AMOC peaks, density in the Labrador-Irminger Sea region reaches a maximum, triggering deep water formation. This density change is caused by a counterclockwise advection of temperature and salinity anomalies at lower latitudes, which we relate to the north-south excursions of the subpolar-subtropical gyre boundary and variations in strength and position of the subpolar gyre and the North Atlantic Current. The AMOC fluctuations are not directly forced by the atmosphere, but occur in a delayed response of the ocean to forcing by the North Atlantic Oscillation, which initiates “intergyre”-gyre fluctuations. Associated with the AMOC is a 60-year sea surface temperature variability in the Atlantic, with a pattern and timescale showing similarities with the real-world Atlantic Multidecadal Variability. This good agreement with observations lends a certain degree of credibility that the mechanism that is described in this article could be seen as representative of the real climate system.
High prevalence of a fungal prion
Debets, A.J.M. ; Dalstra, H.J.P. ; Slakhorst, S.M. ; Koopmanschap-Memelink, A.B. ; Hoekstra, R.F. ; Saupe, S.J. - \ 2012
Proceedings of the National Academy of Sciences of the United States of America 109 (2012)26. - ISSN 0027-8424 - p. 10432 - 10437.
podospora-anserina - vegetative incompatibility - het-s - heterokaryon incompatibility - neurospora-crassa - meiotic drive - yeast prion - mechanism - diseases - genes
Prions are infectious proteins that cause fatal diseases in mammals. Prions have also been found in fungi, but studies on their role in nature are scarce. The proposed biological function of fungal prions is debated and varies from detrimental to benign or even beneficial. [Het-s] is a prion of the fungus Podospora anserina. The het-s locus exists as two antagonistic alleles that constitute an allorecognition system: the het-s allele encoding the protein variant capable of prion formation and the het-S allele encoding a protein variant that cannot form a prion. We document here that het-s alleles, capable of prion formation, are nearly twice as frequent as het-S alleles in a natural population of 112 individuals. Then, we report a 92% prevalence of [Het-s] prion infection among the het-s isolates and find evidence of the role of the [Het-s]/het-S allorecognition system on the incidence of infection by a deleterious senescence plasmid. We explain the het-s/het-S allele ratios by the existence of two selective forces operating at different levels. We propose that during the somatic stage, the role of [Het-s]/HET-S in allorecognition leads to frequency-dependent selection for which an equilibrated frequency would be optimal. However, in the sexual cycle, the [Het-s] prion causes meiotic drive favoring the het-s allele. Our findings indicate that [Het-s] is a selected and, therefore, widespread prion whose activity as selfish genetic element is counteracted by balancing selection for allorecognition polymorphism
Current-Induced Membrane Discharge
Baeko Andersen, M. ; Soestbergen, M. ; Mani, A. ; Bruus, H. ; Biesheuvel, P.M. ; Bazant, M.Z. - \ 2012
Physical Review Letters 109 (2012). - ISSN 0031-9007
ion-exchange membranes - electrochemical thin-films - charge regulation model - concentration polarization - transport phenomena - water dissociation - amphoteric membranes - proton-transfer - electrodialysis - mechanism
Possible mechanisms for overlimiting current (OLC) through aqueous ion-exchange membranes (exceeding diffusion limitation) have been debated for half a century. Flows consistent with electro-osmotic instability have recently been observed in microfluidic experiments, but the existing theory neglects chemical effects and remains to be quantitatively tested. Here, we show that charge regulation and water self-ionization can lead to OLC by “current-induced membrane discharge” (CIMD), even in the absence of fluid flow, in ion-exchange membranes much thicker than the local Debye screening length. Salt depletion leads to a large electric field resulting in a local pH shift within the membrane with the effect that the membrane discharges and loses its ion selectivity. Since salt co-ions, H+ ions, and OH- ions contribute to OLC, CIMD interferes with electrodialysis (salt counterion removal) but could be exploited for current-assisted ion exchange and pH control. CIMD also suppresses the extended space charge that leads to electro-osmotic instability, so it should be reconsidered in both models and experiments on OLC.
Illuminating the off-pathway nature of the molten globule folding intermediate of an a-ß parallel protein
Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Mierlo, C.P.M. van - \ 2012
PLoS ONE 7 (2012)9. - ISSN 1932-6203
azotobacter-vinelandii apoflavodoxin - refractive-index - fluorescence depolarization - spectroscopic ruler - hydrogen-exchange - energy landscape - state - flavodoxin - aggregation - mechanism
Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an a-ß parallel protein
Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1
Sucharitakul, J. ; Wongnate, T. ; Montersino, S. ; Berkel, W.J.H. van; Chaiyen, P. - \ 2012
Biochemistry 51 (2012)21. - ISSN 0006-2960 - p. 4309 - 4321.
para-hydroxybenzoate hydroxylase - biochemical-characterization - pseudomonas-fluorescens - acinetobacter-baumannii - p-hydroxyphenylacetate - genus rhodococcus - mechanism - flavoprotein - purification - degradation
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M–1 s–1. In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s–1. At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is 51 s–1, whereas without 3-HB, the rate constant is 0.43 s–1 at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme–product (2,5-DHB) complex, with a rate constant of 45 ± 2 s–1. The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em0 for the enzyme–substrate complex is -179 ± 1 mV compared to an Em0 of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme–ligand complex to a fully activated form before flavin reduction takes place
CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3
Westra, E.R. ; Erp, P.B.G. ; Künne, T. ; Wong, S.P. ; Staals, R.H.J. ; Seegers, C.L.C. ; Bollen, S. ; Jore, M.M. ; Semenova, E. ; Severinov, K. ; Vos, W.M. de; Dame, R.T. ; Vries, R. de; Brouns, S.J.J. ; Oost, J. van der - \ 2012
Molecular Cell 46 (2012)5. - ISSN 1097-2765 - p. 595 - 605.
rna-polymerase - complex - prokaryotes - mechanism - protein - bacteriophage - resistance - sequence - defense - system
The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization