Short‐term survival and movements of Atlantic sharpnose sharks captured by hook‐and‐line in the north‐east Gulf of Mexico

Ultrasonic telemetry was used to compare post‐release survival and movements of Atlantic sharpnose sharks Rhizoprionodon terraenovae in a coastal area of the north‐east Gulf of Mexico. Ten fish were caught with standardized hook‐and‐line gear during June to October 1999. Atlantic sharpnose sharks were continuously tracked after release for periods of 0·75 to 5·90 h and their positions recorded at a median interval of 9 min. Individual rate of movement was the mean of all distance and time measurements for each fish. Mean ± s.e . individual rate of movement was 0·45 ± 0·06 total lengths per second (L_T s^?1) and ranged from 0·28 to 0·92 L_T s^?1 over all fish. Movement patterns did not differ between jaw and internally hooked Atlantic sharpnose sharks. Individual rate of movement was inversely correlated with bottom water temperature at capture (r² = 0·52, P ≤ 0·05). No consistent direction in movement was detected for Atlantic sharpnose sharks after release, except that they avoided movement towards shallower areas. Capture‐release survival was high (90%), with only one fish not surviving, i.e. this particular fish stopped movement for a period of 10 min. Total rate of movement was total distance over total time (m min^?1) for each Atlantic sharpnose shark. Mean total rate of movement was significantly higher immediately after release at 21·5 m min^?1 over the first 1·5 h of tracking, then decreased to 11·2 m min^?1 over 1·5–6 h, and 7·7 m min^?1 over 3–6 h (P ≤ 0·002), which suggested initial post‐release stress but quick recovery from capture. Thus, high survival (90%) and quick recovery indicate that the practice of catch‐and‐release would be a viable method to reduce capture mortality for R. terraenovae.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

Characterization of an L-arabinose isomerase from Bacillus subtilis

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k _cat of 14,504 min⁻¹ and a k _cat/K _m of 121 min⁻¹ mM⁻¹ for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.     

Biosorption of Malathion using dry cells of Bacillus species S14 in a packed bed column reactor

Abstract

The ability of dried bacterial strain Bacillus sp. S₁₄ to adsorb Malathion in a packed bed column reactor was studied. The effects of important design parameters such as bed height, flow rate and influent Malathion concentration on Malathion removal from an aqueous solution was studied using a packed bed column reactor. The optimised conditions for maximum Malathion removal were found to be: flow rate: 5 mL min^-1, bed height: 6.0 cm and influent Malathion concentration: 25 mg L^-1. The Adams-Bohart model, Wolborska model, Thomas model, Yoon and Nelson Model were employed to determine characteristic parameters such as N₀ (saturation concentration, mg L^-1), β_o (external mass transfer coefficient, min^-1), k _Th(Thomas rate constant, mL min^-1mg^-1), q₀ (maximum solid phase concentration of the solute, mg L^-1), k_YN (rate constant, min^-1) and τ (time required for 50 % adsorbate breakthrough time, min) which are useful for process design. Data were fitted with Adams-Bohart model at lower region of (C/C₀) values but more accurately fitted with Wolborska and Thomas model.     

Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones

Molecular chaperones are ATP‐consuming machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly. GroEL is an all‐purpose stochastic machine that assists misfolded substrate proteins to fold. The RNA chaperones such as CYT‐19, which are ATP‐consuming enzymes, help the folding of ribozymes that get trapped in metastable states for long times. GroEL does not interact with the folded proteins but CYT‐19 disrupts both the folded and misfolded ribozymes. The structures of GroEL and RNA chaperones are strikingly different. Despite these differences, the iterative annealing mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, T (apo), R (ATP bound), and R^″ (ADP bound). Analyses of the experimental data show that the efficiency of the GroEL–GroES machinery and mutants is determined by the resetting rate k _{R ″ → T} , which is largest for the wild‐type (WT) GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady‐state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

Microsecond simulations of the folding/unfolding thermodynamics of the Trp‐cage miniprotein

We study the unbiased folding/unfolding thermodynamics of the Trp‐cage miniprotein using detailed molecular dynamics simulations of an all‐atom model of the protein in explicit solvent using the Amberff99SB force field. Replica‐exchange molecular dynamics simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs/replica timescale. The total simulation time used in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P, T), over the whole region of temperatures and pressures sampled in the simulations. The ΔG(P, T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure‐induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (T_f = 321 K), free energy, and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semiempirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Molecular dynamics simulations of certain mutant peptide models from staphylococcal nuclease reveal that initial hydrophobic collapse associated with turn propensity drives β‐hairpin folding

An important nucleation event during the folding of staphylococcal nuclease involves the formation of a β‐hairpin by the sequence ²¹DTVKLMYKGQPMTFR³⁵. Earlier studies show that the turn sequence ‘YKGQP’ has an important role in the folding of this β‐hairpin. To understand the active or passive nature of the turn sequence ‘YKGQP’ in the folding of the aforementioned β‐hairpin sequence, we studied glycine mutant peptides Ac‐²DTVKLMYGGQPMTFR¹⁶‐NMe (K9G:15), Ac‐²DTVKLMYKGGPMTFR¹⁶‐NMe (Q11G:15), Ac‐²DTVKLMYGGGPMTFR¹⁶‐NMe (K9G/Q11G:15), and Ac‐²DTVKLMGGGGGMTFR¹⁶‐NMe (penta‐G:15) by using molecular dynamics simulations, starting with two different unfolded states, polyproline II and extended conformational forms. Further, 5mer mutant turn peptides Ac‐²YGGQP⁶‐NMe (K3G:5), Ac‐²YKGGP⁶‐NMe (Q5G:5), Ac‐²YGGGP⁶‐NMe (K3G/Q5G:5), and Ac‐²GGGGG⁶‐NMe (penta‐G:5) were also studied individually. Our results show that an initial hydrophobic collapse and loop closure occurs in all 15mer mutants, but only K9G:15 mutant forms a stable native‐like β‐hairpin. In the other 15mer mutants, the hydrophobic collapsed state would not proceed to β‐hairpin formation. Of the different simulations performed for the penta‐G:15 mutant, in only one simulation a nonnative β‐hairpin conformation is sampled with highly flexible loop region (⁸GGGGG¹²), which has no specific conformational preference as a 5mer. While the sequence ‘YGGQP’ in the K3G:5 simulation shows relatively higher β‐turn propensity, the presence of this sequence in K9G:15 peptide seems to be driving the β‐hairpin formation. Thus, these results seem to suggest that for the formation of a stable β‐hairpin, the initial hydrophobic collapse is to be assisted by a turn propensity. Initial hydrophobic collapse alone is not sufficient to guide β‐hairpin formation. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Contributions of water transfer energy to protein‐ligand association and dissociation barriers: Watermap analysis of a series of p38α MAP kinase inhibitors

In our previous work, we proposed that desolvation and resolvation of the binding sites of proteins can serve as the slowest steps during ligand association and dissociation, respectively, and tested this hypothesis on two protein‐ligand systems with known binding kinetics behavior. In the present work, we test this hypothesis on another kinetically‐determined protein‐ligand system—that of p38α and eight Type II BIRB 796 inhibitor analogs. The k_on values among the inhibitor analogs are narrowly distributed (10⁴ ≤ k_on ≤ 10⁵ M^?1 s^?1), suggesting a common rate‐determining step, whereas the k_off values are widely distributed (10^?1 ≤ k_off ≤ 10^?6 s^?1), suggesting a spectrum of rate‐determining steps. We calculated the solvation properties of the DFG‐out protein conformation using an explicit solvent molecular dynamics simulation and thermodynamic analysis method implemented in WaterMap to predict the enthalpic and entropic costs of water transfer to and from bulk solvent incurred upon association and dissociation of each inhibitor. The results suggest that the rate‐determining step for association consists of the transfer of a common set of enthalpically favorable solvating water molecules from the binding site to bulk solvent. The rate‐determining step for inhibitor dissociation consists of the transfer of water from bulk solvent to specific binding site positions that are unfavorably solvated in the apo protein, and evacuated during ligand association. Different sets of unfavorable solvation are evacuated by each ligand, and the observed dissociation barriers are qualitatively consistent with the calculated solvation free energies of those sets.     

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

After cultivation on (R,S)‐2‐(2,4‐dichlorophenoxy)propionate, two α‐ketoglutarate‐dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R‐enantiomer of substituted phenoxypropionate derivatives: the K_m values were 55 μM and 30 μM, the k_cat values 55 min^–1 and 34 min^–1 with (R)‐2‐(2,4‐dichlorophenoxy)propionate [(R)‐2,4‐DP] and (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S‐enantiomers with K_m values of 49 μM and 22 μM, and k_cat values of 50 min^–1 and 46 min^–1 with (S)‐2‐(2,4‐dichlorophenoxy)propionate [(S)‐2,4‐DP] and (S)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K_m = 123 μM, k_cat = 36 min^–1) with significant activity. As the second substrate, only α‐ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K_i values of 3 mM with (R)‐2,4‐DP and 1.5 mM with (S)‐2,4‐DP. The reaction was strictly dependent on the presence of Fe²⁺ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).     

Oxygen transfer in Streptomyces fermentation broths

The oxygen transfer coefficient has been investigated in S. noursci and S. lavendulae fermentation broths obtained from fermentors of different operating volumes (61., 30001., 20,0001.). Fermentors had K_La_s values ranging from 1.0 to 17.0 min^?1, calculated from sulphite oxidation rates. The dynamic measurement of the volumetric oxygen transfer coefficient. (K_La) has been performed in the different fermenting systems. As the fermentation progressed, especially in the first stages, K_La values have decreased in both fermentations and in each system of fermentors. In order to characterise the whole fermenting system an average K_La_s was calculated from the obtained K_La values. The average K_La grew with increasing K_La_s values and ranged from 0.03 to 3.72 min^?l. Some factors possibly having an influence on the, change of K_La have been studied. The oxygen transfer coefficients of the broths have been measured in falling films and ranged from 0.05 to 0.4 cm min^?1. The flow conditions have been characterized by Reynolds numbers of broths varying between 1.0 and 60.0. The average thickness of the falling films have been measured and plotted against Reynolds number. The Re⁺ which is the breaking-point of the plot increased as the fermentation proceeded. In the region of Re⁺ the values of the oxygen transfer coefficient increased rapidly. An approximate correlation could be established between the Re⁺ and the physical properties of fermentation broth.     

Transpiration rate relates to within‐ and across‐species variations in effective path length in a leaf water model of oxygen isotope enrichment

Stable oxygen isotope ratio of leaf water (δ¹⁸O_L) yields valuable information on many aspects of plant–environment interactions. However, current understanding of the mechanistic controls on δ¹⁸O_L does not provide complete characterization of effective path length (L) of the Péclet effect, – a key component of the leaf water model. In this study, we collected diurnal and seasonal series of leaf water enrichment and estimated L in six field‐grown angiosperm and gymnosperm tree species. Our results suggest a pivotal role of leaf transpiration rate (E) in driving both within‐ and across‐species variations in L. Our observation of the common presence of an inverse scaling of L with E in the different species therefore cautions against (1) the conventional treatment of L as a species‐specific constant in leaf water or cellulose isotope (δ¹⁸O_p) modelling; and (2) the use of δ¹⁸O_p as a proxy for g_s or E under low E conditions. Further, we show that incorporation of a multi‐species L‐E scaling into the leaf water model has the potential to both improve the prediction accuracy and simplify parameterization of the model when compared with the conventional approach. This has important implications for future modelling of oxygen isotope ratios.     

Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Loop dependence of the stability and dynamics of nucleic acid hairpins

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as ~L^{8.5 ± 0.5}, in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl₂, the stabilities of both ssDNA and RNA hairpins scale as ~L^{4 ± 0.5}, indicating that the intraloop interactions are weaker in the presence of Mg²⁺. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl₂) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, ~L^{2.2 ± 0.5} and ~L^{2.6 ± 0.5}, respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na⁺ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.