Evolutionary trajectory of fish Piscine novirhabdovirus (=Viral Hemorrhagic Septicemia Virus) across its Laurentian Great Lakes history: Spatial and temporal diversification

Piscine novirhabdovirus = Viral Hemorrhagic Septicemia Virus (VHSV) first appeared in the Laurentian Great Lakes with large outbreaks from 2005 to 2006, as a new and novel RNA rhabdovirus subgenogroup (IVb) that killed >30 fish species. Interlude periods punctuated smaller more localized outbreaks in 2007, 2010, and 2017, although some fishes tested positive in the intervals. There have not been reports of outbreaks or positives from 2018, 2019, or 2020. Here, we employ a combined population genetics and phylogenetic approach to evaluate spatial and temporal evolutionary trajectory on its G‐gene sequence variation, in comparison with whole‐genome sequences (11,083 bp) from a subset of 44 individual isolates (including 40 newly sequenced ones). Our results show that IVb (N = 184 individual fish isolates) diversified into 36 G‐gene haplotypes from 2003 to 2017, stemming from two originals (“a” and “b”). G‐gene haplotypes “a” and “b” differed by just one synonymous single‐nucleotide polymorphism (SNP) substitution, remained the most abundant until 2011, then disappeared. Group “a” descendants (14 haplotypes) remained most prevalent in the Upper and Central Great Lakes, with eight (51%) having nonsynonymous substitutions. Group “b” descendants primarily have occurred in the Lower Great Lakes, including 22 haplotypes, of which 15 (68%) contained nonsynonymous changes. Evolutionary patterns of the whole‐genome sequences (which had 34 haplotypes among 44 isolates) appear congruent with those from the G‐gene. Virus populations significantly diverged among the Upper, Central, and Lower Great Lakes, diversifying over time. Spatial divergence was apparent in the overall patterns of nucleotide substitutions, while amino acid changes increased temporally. VHSV‐IVb thus significantly differentiated across its less than two decades in the Great Lakes, accompanied by declining outbreaks and virulence. Continuing diversification likely allowed the virus to persist at low levels in resident fish populations, and may facilitate its potential for further and future spread to new habitats and nonacclimated hosts.     

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site‐directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k _cat relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k _cat/K _M across the series. Only modest decreases in k _cat/K _M are observed for the E85Q variants with little effect on k _cat. Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst.     

THE SOLUBILITY OF TYROSINE IN ACID AND IN ALKALI

Measurements have been made of the solubility at 25°C. of tyrosine in hydrochloric acid and in sodium hydroxide solutions varying from 0.001 to 0.05 M, and also in distilled water. The pH of the saturated solutions was measured with the hydrogen electrode. The following values for the ionization constants of tyrosine have been obtained from the measurements: k_b = 1.57 x 10^–12, k_a1 = 7.8 x 10^–10, k_a2 = 8.5 x 10^–11. The changes in solubility with pH can be satisfactorily explained by the use of these ionization constants.     

A Highly Efficient Recombinant Laccase from the Yeast Yarrowia lipolytica and Its Application in the Hydrolysis of Biomass

A modified thermal asymmetric interlaced polymerase chain reaction was performed to obtain the first yeast laccase gene (YlLac) from the isolated yeast Yarrowia lipolytica. The 1557-bp full-length cDNA of YlLac encoded a mature laccase protein containing 519 amino acids preceded by a signal peptide of 19 amino acids, and the YlLac gene was expressed in the yeast Pichia pastoris. YlLac is a monomeric glycoprotein with a molecular mass of ~55 kDa as determined by polyacrylamide-gel electrophoresis. It showed a higher catalytic efficiency towards 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (k_cat/K_m = 17.5 s^-1 μM^-1) and 2,6-dimethoxyphenol (k_cat/K_m = 16.1 s^-1 μM^-1) than other reported laccases. The standard redox potential of the T1 site of the enzyme was found to be 772 mV. The highest catalytic efficiency of the yeast recombinant laccase, YlLac, makes it a good candidate for industrial applications: it removes phenolic compounds in acid-pretreated woody biomass (Populus balsamifera) and enhanced saccharification.     

Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C^pro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C^pro, we measured cleavage kinetics of predicted SVV-001 3C^pro substrates. An efficient substrate, L/VP4 (k_cat/K_M = 1932 ± 183 M^-1s^-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (k_cat/K_M = 17446 ± 2203 M^-1s^-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C^pro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C^pro, with direct implications for anti-cancer therapeutic development.     

OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat correlates (R² = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved K_M, k_cat, and k_cat/K_M for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S.     

Characterization and comparison of arcelin seed protein variants from common bean

Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L., were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and M_rs were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M_rs, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins-3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-1d and -2) could be distinguished by M_rs and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross-reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-1t and arcelin-4 also could be distinguished from each other based on M_rs and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean α-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family.     

A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K_m = 6.2 mM; k_cat = 5.76 s⁻¹) and glutaramic acid (K_m = 2.8 mM; k_cat = 2.23 s⁻¹). However, the substrates of known amidases such as short-chain (C₂ to C₄) aliphatic amides, long-chain (above C₁₆) aliphatic amides, amino acid amides, aliphatic diamides, α-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M_r of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv)

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus that causes one of the most important finfish diseases, affecting over 70 marine and freshwater species. It was discovered in European cultured fish in 1938 and since has been described across the Northern Hemisphere. Four strains and several substrains have been hypothesized, whose phylogenetic relationships and evolutionary radiation are evaluated here in light of a quasispecies model, including an in-depth analysis of the novel and especially virulent new substrain (IVb) that first appeared in the North American Laurentian Great Lakes in 2003. We analyze the evolutionary patterns, genetic diversity, and biogeography of VHSv using all available RNA sequences from the glycoprotein (G), nucleoprotein (N), and non-virion (Nv) genes, with Maximum Likelihood and bayesian approaches. Results indicate that the G gene evolves at an estimated rate of μ=2.58×10(-4) nucleotide substitutions per site per year, the N gene at μ=4.26×10(-4), and Nv fastest at μ=1.25×10(-3). Phylogenetic trees from the three genes largely are congruent, distinguishing strains I-IV as reciprocally monophyletic with high bootstrap and posterior probability support. VHSv appears to have originated from a marine ancestor in the North Atlantic Ocean, diverging into two primary clades: strain IV in North America (the Northwestern Atlantic Ocean), and strains I-III in the Northeastern Atlantic region (Europe). Strain II may comprise the basal group of the latter clade and diverged in Baltic Sea estuarine waters; strains I and III appear to be sister groups (according to the G and Nv genes), with the former mostly in European freshwaters and the latter in North Sea marine/estuarine waters. Strain IV is differentiated into three monophyletic substrains, with IVa infecting Northeastern Pacific salmonids and many marine fishes (with 44 unique G gene haplotypes), IVb endemic to the freshwater Great Lakes (11 haplotypes), and a newly-designated IVc in marine/estuarine North Atlantic waters (five haplotypes). Two separate substrains independently appeared in the Northwestern Pacific region (Asia) in 1996, with Ib originating from the west and IVa from the east. Our results depict an evolutionary history of relatively rapid population diversifications in star-like patterns, following a quasispecies model. This study provides a baseline for future tracking of VHSv spread and interpreting its evolutionary diversification pathways.     

Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA

Cercospora leaf spot, caused by the hemibiotrophic fungal pathogen Cercospora beticola, is the most economically damaging foliar disease of sugarbeet worldwide. Although most C. beticola populations display characteristics reminiscent of sexual recombination, no teleomorph has been described. To assess whether populations in northern United States have characteristics consistent with sexual reproduction, 1024 isolates collected over a 3-y period were analyzed for frequency and distribution of mating type genes. After clone correction, an approximately equal distribution of mating types was found for each sampling year. Mating type frequency was also assessed in individual lesions. Lesions always consisted of isolates with a single mating type and microsatellite haplotype, but both mating types and up to five microsatellite haplotypes could be found on an individual leaf. The MAT1-1-1 and MAT1-2-1 genes were sequenced from 28 MAT1-1 and 28 MAT1-2 isolates, respectively. Three MAT1-1-1 nucleotide haplotypes were identified that encoded a single amino acid sequence. For MAT1-2-1, five nucleotide haplotypes were identified that encoded four protein variants. MAT1-1-1 and MAT1-2-1 gene expression analyses were conducted on plants inoculated with either or both mating types. MAT1-1-1 expression remained low, but MAT1-2-1 spiked during late stages of colonization. A segment of the MAT1-2-1 coding sequence was also found in MAT1-1 isolates. Taken together, these results suggest that C. beticola has the potential for sexual reproduction.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS : II. THE ACTION OF SUBSTITUTED BENZOIC ACIDS AND OF BENZOIC AND SALICYLIC ACIDS AS INFLUENCED BY THEIR SALTS.

1. Comparison of the rates of activation of unfertilized starfish eggs in pure solutions of a variety of parthenogenetically effective organic acids (fatty acids, carbonic acid, benzoic and salicylic acids, chloro- and nitrobenzoic acids) shows that solutions which activate the eggs at the same rate, although widely different in molecular concentration, tend to be closely similar in C_H. The dissociation constants of these acids range from 3.2 x 10^–7 to 1.32 x 10^–3. 2. In the case of each of the fourteen acids showing parthenogenetic action the rate of activation (within the favorable range of concentration) proved nearly proportional to the concentration of acid. The estimated C_H of solutions exhibiting an optimum action with exposures of 10 minutes (at 20°) lay typically between 1.1 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.96), and in most cases between 1.6 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.8). Formic acid (C_H = 4.2 x 10^–4 M) and o-chlorobenzoic acid (C_H = 3.5 x 10^–4 M) are exceptions; o-nitrobenzoic acid is ineffective, apparently because of slow penetration. 3. Activation is not dependent on the penetration of H ions into the egg from without, as is shown by the effects following the addition of its Na salt to the solution of the activating acid (acetic, benzoic, salicylic). The rate of activation is increased by such addition, to a degree indicating that the parthenogenetically effective component of the external solution is the undissociated free acid. Apparently the undissociated molecules alone penetrate the egg freely. It is assumed that, having penetrated, they dissociate in the interior of the egg, furnishing there the H ions which effect activation. 4. Attention is drawn to certain parallels between the physiological conditions controlling activation in the starfish egg and in the vertebrate respiratory center.     

Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon–carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest k_cat (> 10^− 3 s^− 1) and k_cat/K_M (11–25 M^− 1 s^− 1) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the > 10⁵-fold rate accelerations that were achieved are within 1–3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (k_cat/k_uncat = 10⁶ to 10⁸) and an extensively evolved computational design (k_cat/k_uncat > 10⁷). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9₂₅-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9₂₅-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5′-product terminating in a 2′,3′-phosphodiester and a 3′-product terminating in a 5′-OH. Under single turnover conditions at 24°C, 9₂₅-11t displays a maximum first-order rate constant, k_cat, of 0.037 min⁻¹ and a catalytic efficiency, k_cat/K_m, of 5.3 × 10⁵ M⁻¹ min⁻¹. The measured value of k_cat under catalyst excess conditions agrees with the value of k_cat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9₂₅-11t was gauged in terms of k_cat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k_cat values by a factor of 4 to 250, indicating that 9₂₅-11t displays significant sequence specificity despite the lack of an apparent Watson–Crick base-pairing scheme for recognition.     

Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus sp.

Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O₂ to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn⁷⁵, Asn²³⁸, and Asn⁴⁵⁸ were elucidated. Initial rate kinetic analysis revealed that the k _cat, K _m, and k _cat/K _m of nLcc4 with substrate ABTS were 3,382 s ^-1, 65.0 ± 6.5 μM, and 52 s ^-1μM^-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s ^-1, 56.7 ± 3.2 μM, and 0.004 s ^-1μM^-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.     

A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids

Leukotriene A₄ hydrolase (LTA4H––EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA₄ through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k _cat/K _m values). Among them, the benzyl ester of aspartic acid exhibited a k _cat/K _m value that was more than two orders of magnitude higher (1.75 × 10⁵ M^?1 s^?1) as compared to l-Arg (1.5 × 10³ M^?1 s^?1). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.     

Two Homologous EF-G Proteins from Pseudomonas aeruginosa Exhibit Distinct Functions

Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K _M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k _cat) for the hydrolysis of GTP than EF-G1A; 0.2 s^-1 vs. 0.04 s^-1. These values resulted in specificity constants (k _cat ^obs/K _M) for EF-G1A and EF-G1B of 0.5 x 10³ s^-1 M^-1 and 3.0 x 10³ s^-1 M^-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.     

Synonymous Rare Arginine Codons and tRNA Abundance Affect Protein Production and Quality of TEV Protease Variant

It has been identified that a TEV protease (TEVp) variant, TEVp^5M, displays improved solubility. Here, we constructed fifteen TEVp^5M variants with one or more of six rare arginine codons in the coding sequence replaced with abundant E. coli arginine codons. These codon variants expressed in either E. coli BL21 (DE3) or Rossetta (DE3) showed different solubility and activity. Supply of rare tRNAs increased the tendency of certain codon variants to form insoluble aggregates at early induction stage, as determined by the fused S-tag. About 32% increase in soluble protein production of M5 variant with four synonymously mutated arginine codons was identified in Rossetta (DE3) cells using GFP fusion reporter, comparable to that of TEVp^5M. After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp^5M on cleaving the native or modified recognition sequence incorporated between GST and E. coli diaminopropionate ammonialyase by enzyme-coupled assay, whereas purified M5 variant showed activity similar to the TEVp^5M. Supply of rare tRNAs caused the decrease of activity of TEVp^5M and M5 by about 21%. Our results revealed that engineering of highly soluble TEVp variants can be achieved by the combined mutations of amino acid residues and optimization of specific rare codons, whereas simple augment of rare tRNAs abundance resulted in partial loss of activity.     

Conformational Heterogeneity of Cyclosporin A in Cyclophilin 18 Binding

The immunosuppressive drug cyclosporin A (CsA) binds to its receptor protein cyclophilin 18 (Cyp18) in two distinct kinetic phases, while the mechanism remains elusive. Stopped-flow measurements coupled with titration and competition experiments were used to investigate the puzzling two-phase process of CsA and Cyp18 interaction. This study leads to the dissection of different conformational fractions of either direct fast binding or slow binding with rate-limiting conformational inter-conversion and the real-time measurement of k_on value (8.34 ± 0.22 x10⁶ M^-1s^-1) in solution. Furthermore, our study indicates that the structure of CsA during dissociation from the protein possesses a distribution of conformations different from those in solution under equilibrium condition.