The influence of increasing chlorine content on the accumulation and metabolism of polychlorinated biphenyls (PCBs) by Paul's Scarlet Rose cells

Four radiolabled congeners of biphenyls with increasing chlorine content (biphenyl; 1-monochlorobiphenyl; 2,2,4,4-tetrachlorobiphenyl; and 2,2,4,4,5,5-hexachlorobiphenyl) were provided to suspension cultures of rose (Rosa sp. cv. Paul's Scarlet) for 4 days. Both the kinetics of ¹⁴C exchange between the cells and medium, and the metabolism of the parent compounds depended on the chlorine content of the congeners. Analysis of both the cells and their medium showed that of the recovered radioactivity 88%, 86%, and 3% of the biphenyl, 1-PCB, and 2,2,4,4-PCB were metabolized respectively to polar and insoluble residue products. The 2,2,4,4,5,5-PCB did not appear to be metabolized.     

Cannabis 25 1, biotransformation of cannabidiol and cannabidiolic acid by Pinellia ternata tissue segments

Two marihuana constituents, cannabidiol (1) and cannabidiolic acid (4) were each converted mainly to two metabolites using tissue segments of Pinellia ternata tuber. The structures of the metabolites formed from 1 were determined to be 1-O-D-glucopyranoside and 1-O-D-diglucopyranoside by ¹H nmr, ¹³C nmr and fabms. Those from 4 were determined as 4-O-D-glucopyranoside and 10-hydroxyl 4-O-D-glucopyranoside. In time course experiments, 1 was absorbed rapidly by the tissues and glucosylated. Hydroxylation subsequent to the glucosylation occurred at the pentyl group in 4.1 For Part 24, see Taura F, Morimoto S, Shoyama Y, Mechoulam R (1995) J Am Chem Soc 117:9766–9767     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-beta 33-53).

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH- chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH--(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel -pleated sheet, turns including a -turn, other structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH--(33–53). These features included an antiparallel -sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a -turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (70%) over thecis (30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH--(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel -sheet and -turn which fold to produce a distorted hairpin. The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.Abreviations used: hFSH, human follicle-stimulating hormone; PB, 25 mM Na₂KPO₄, 25 mM KH₂PO₄, and 5 mM Mg Cl₂; CD, circular dichroism spectrapolarimetry; NMR, nuclear magnetic resonance spectrometry; COSY, homonuclear correlated spectroscopy; NOESY, 2D nuclear Overhauser effect spectroscopy; HOHAHA, homonuclear Hartman-Han coherence transfer; HMQCHY, reverse-detected heteronuclear multiple shift correlation, one bond; HMBC, reverse-detected heteronuclear multiple bond correlation; S/N, signal to noise ratio; TFE, trifluoroethanol.Dr. Santa-Coloma is on leave of absence from the National Research Council of Argentina (CONICET).     

The three-dimensional structure of acyl-coenzyme A binding protein from bovine liver: Structural refinement using heteronuclear multidimensional NMR spectroscopy

Summary The 3D structure of bovine recombinant acyl-coenzyme A binding protein has been determined using multidimensional heteronuclear magnetic resonance spectroscopy in a study that combines investigations of ¹⁵N-labeled and unlabeled protein. The present structure determination is a refinement of the structure previously determined (Andersen, K.V. and Poulsen, F.M. (1992) J. Mol. Biol., 226, 1131–1141). It is based on 1096 distance restraints and 124 dihedral angle restraints of which 69 are for -angles and 8 for chiral centers and 47 for prochiral centers. The new experimental input for the structure determination has provided an increase of 263 distance restraints, 5 -angle restraints, and 32 -angle restraints in 2 chiral centers, and 31 prochiral centers restraining an additional 23 ¹, 8 ², and 1 ³ angles. The increase of 300 distance and dihedral angle restraints representing an additional 30% of input parameters for the structure determination has been shown to be in agreement with the first structure. A set of 29 structures has been calculated and each of the structures has been compared to a mean structure to give an atomic root mean square deviation of 0.44±0.12 (1 is 0.1 nm) for the backbone atoms C, C, and N in the four -helices A1, residues 4–15, A2, residues 21–36, A3, residues 51–62 and A4, residues 65–84. The loop-region of residues Gly⁴⁵-Lys⁵⁰ could not be defined by the restraints obtained by NMR.The program PRONTO has been used for the spectrum analysis, assignment of the individual nuclear Overhauser effects, the integration of the cross peaks, and the measurement of the coupling constants. The programs DIANA, X-PLOR and INSIGHT have been used in the structure calculations and evaluations.     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.     

Induction of antifreeze protein production by juvenile hormone in larvae of the beetle,Dendroides canadensis

Summary Larvae of the beetleDendroides canadensis accumulate protein antifreezes during the winter.D. canadensis which were collected in the early fall, prior to the initiation of cold hardening processes, were treated with either 3.3 or 6.6 g juvenile hormone I topically in acetone and maintained for 21 days under normally non-inductive acclimation conditions (16 light/8 dark, 20 °C). Hormone treated animals significantly elevated the levels of antifreeze protein in their hemolymph compared to those of acetone treated and untreated controls or animals measured on the day of collection. D. canadensis treated with the anti-JH compound precocene II (P2) in acetone for 24 h at a concentration of 20 g/cm² (a dose below LD₅₀ for behavioral survival) and then maintained under acclimation conditions conducive to antifreeze protein production (8 light/16 dark, 20 °C) for 2 weeks failed to elevate levels of antifreeze. Acetone treated control animals accumulated a significant concentration of antifreeze protein.D. canadensis were also treated with 20 and 150 g/cm² P2 (a dose below the LD₅₀ for gross survival) followed by acclimation to short (8 h) photoperiod at 10 °C. All animals receiving the higher P2 dosage failed to elevate antifreezes while only 42.9% of the individuals treated with the lower dosage initiated antifreeze protein production. In contrast, over 80% of untreated and 70% of acetone treated controls responded to these inductive acclimation conditions by elevating antifreeze concentrations.These results indicate that juvenile hormone participates in the seasonal control of antifreeze protein production inDendroides canadensis. Since this species does not enter a diapause state prior to or throughout the winter this is the first evidence establishing a direct hormonal mechanism involved with insect cold hardiness.     

Investigations of the pigments fromCytophaga johnsonae Cy j1

Besides carotenoids a complex of flexirubin-type pigments was isolated from the gliding bacteriumCytophaga johnsonae Cy j1 and separated into 6 components, which partly containe chlorine. In spite of the fact that these components still consist of pigment mixtures, the gross structures of 18 new flexirubin-type pigments could be deduced by spectroscopic and chemical investigations. The results open insights into biosynthesis and structural variety of the flexirubins, the novel non-isoprenoid pigments recently found inFlexibacter elegans.Non-Standard Abbreviations FT Fourier transformation - HPLC high pressure liquid chromatography - M⁺ molecular ion - M/M resolution of mass spectrometer - mu mass units - t _R retention time - TLC thin layer chromatography Part 16: Investigations on metabolites of microorganisms. Part. XV: H. Achenbach, W. Kohl, H. Reichenbach: Die Hauptpigmente vonCytophaga johnsonae. Tetrahedron Lett.1977, 1061. Part XIV: H. Achenbach, J. Witzke: Totalsynthese des Flexirubindimethylethers. Angew. Chem.89, 198 (1977); Angew. Chem. Int. Ed. Engl.16, 191 (1977)     

Variable generation times and Darwinian fitness measures

Summary Reproductive value (RV) and net reproductive output (R _o) are frequently used fitness measures. We argue that they are only appropriate when intervals between reproductive events are fixed, as they are dimensionless generation-to-generation scalings with units offspring per parent. A fitness measure should account for two different effects of a decrease in generation time: (1) increased survival due to shorter exposure to mortality agents and (2) increased frequency of reproduction.R _o andRV deal with the first of these two effects, while a measure with a physical dimensionper time [T^–1] is needed to account for the second. The Malthusian growth parameter,r, meets this requirement and in situations where time to reproduction is variable, we propose, the instantaneous rate of spread of descendants (from an individual) be used instead ofR _o. As an alternative toRV, we suggest using the instantaneous difference = –r, wherer is the population rate of increase. WhileRV andR _o are dimensionless ratios, , and areper time rates which are appropriate in accounting for alterations in generation time.     

Refined solution structure of the dimeric N-terminal HHCC domain of HIV-2 integrase

The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1–55, named IN_1–55) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739–746], consists of four -helices and is well defined. Helices 1, 2 and 3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix 3. The orientation of the two monomeric units with respect to each other shows considerable variation. ¹⁵N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN_1–55 shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN_1–55 [Cai et al. (1997) Nat. Struct. Biol., 4, 567–577], where the corresponding residues are positively charged and neutral ones.     

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

Summary We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with ¹⁵N. The requirement of ¹³C-/¹⁵N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., _0c~1). The technique was applied to an unstructured ¹⁵N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s^-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.     

Asparagine and glutamine side-chain conformation in solution and crystal: A comparison for hen egg-white lysozyme using residual dipolar ouplings

Experimental ¹⁵N–¹H and ¹H–¹H residual dipolar couplings (RDCs) for the asparagine (Asn) and glutamine (Gln) side chains of hen egg-white lysozyme are measured and analysed in conjunction with ¹N relaxation data, information about ¹ torsion angles in solution and molecular dynamics simulations. The RDCs are compared to values predicted from 16 high-resolution crystal structures. Two distinct groups of Asn and Gln side chains are identified. The first contains residues whose side chains show a fixed, relatively rigid, conformation in solution. For these residues there is good agreement between the experimental and predicted RDCs. This agreement improves when the experimental order parameter, S, is included in the calculation of the RDCs from the crystal structures. The comparison of the experimental RDCs with values calculated from the X-ray structures shows that the similarity between the oxygen and nitrogen electron densities is a limitation to the correct assignment of the Asn and Gln side-chain orientation in X-ray structures. In the majority of X-ray structures a 180° rotation about ² or ³, leading to the swapping of N ² and O ¹, is necessary for at least one Asn or Gln residue in order to achieve good agreement between experimental and predicted RDCs. The second group contains residues whose side chains do not adopt a single, well-defined, conformation in solution. These residues do not show a correlation between the experimental and predicted RDCs. In many cases the family of crystal structures shows a range of orientations for these side chains, but in others the crystal structures show a well-defined side-chain position. In the latter case, this is found to arise from crystallographic contacts and does not represent the behaviour of the side chain in solution.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

A critical evaluation of the predicted and X-ray structures of alpha-lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine -Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon -Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C of residues 1–95, 1.1 Å).     

Accuracy of Secondary Structure and Solvent Accessibility Predictions for a Clostridial Neurotoxin C-Fragment

Earlier studies used Rost and Sander's artificial neural network [(1993a), J. Mol. Biol. 232, 584–599] to predict the secondary structures [Lebeda and Olson (1994), Proteins 20, 293–300] and residue solvent accessibilities [Lebeda and Olson (1997), J. Protein Chem. 16, 607–618] of the clostridial neurotoxins. Because the X-ray crystal structure of the 50-kDa C-terminal half of the heavy chain of tetanus toxin was recently determined, this report evaluates the accuracy of these network-derived predictions. For this predominantly -strand-containing fragment, predictions, on a per-residue basis, for both secondary structure and solvent accessibility were about 70% accurate. A more flexible and realistic analysis based on overlapping segments yielded accuracies of over 80% for the three-state secondary structure and for the two-state accessibility predictions. Because the accuracies of these predictions are comparable to those made by Rost and Sander using a dataset of 126 nonhomologous globular proteins, our predictions provide a quantitative foundation for gauging the results when building by homology the structures of related proteins.     

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem. 265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem. 189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.     

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.     

The role of an active-site lysyl residue of spinach phosphoribulokinase as explored by site-directed mutagenesis

Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycle enzyme phosphoribulokinase (PRK) from spinach has been assigned to the active-site region [Miziorkoet al. (1990),J. Biol. Chem. 265, 3642–3647]. The equivalent position is occupied by lysyl or arginyl residues in the PRK from both prokaryotic and eukaryotic sources, suggesting a requirement for a basic residue at this location. To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl, glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesis. All of the mutant enzymes retain substantial kinase activity; and even in the case of the radical substitution by glutamate, theK _m values for ATP and ribulose 5-phosphate are not perturbed significantly. Glutamate at position-68 may destabilize tertiary structure, because the yield of this mutant protein from transformedE. coli is quite low compared to that of the other proteins in this series. Despite the active-site proximity of Lys68, our results show that this residue does not play a key role in catalysis or substrate binding.     

Backbone dynamics of ribonuclease T1 and its complex with 2′GMP studied by two-dimensional heteronuclear NMR spectroscopy

Summary The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2GMP have been studied by ¹⁵N longitudinal and transverse relaxation experiments, combined with {¹H, ¹⁵H} NOE measurements. The intensity decay of individual amide cross peaks in a series of (¹H, ¹⁵N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972–8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359–375) was fitted to a single exponential by using a simplex algorithm in order to obtain ¹⁵N T₁ and T₂ relaxation times. These experimentally obtained values were analysed in terms of the model-free approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559; 4559–4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.     

Comparisons of creatine kinase primary structures

Comparisons of nine creatine kinase sequences show that 67% of the protein sequence is identical among rabbit, rat, mouse, and chicken muscle, rabbit, rat, and chicken brain, and electric organ sequences from two species of electric ray(Torpedo). The extensive homology precludes a facile prediction of active-site residues based on sequence conservation. The sequences are more similar within isozyme types than are the different isozymes from any one species. There are 35 positions in the muscle and brain sequence pairs for three species which differentiate the two forms. TheTorpedo sequences do not fall completely into either of these patterns. Except for homology with partial sequences of other ATP-guanidino phosphotransferases, no significant homology with other protein or nucleic acid sequences in available databases was found. Preliminary secondary structural predictions suggest that the C-terminal half of the protein is likely an /-type protein. Placement in the sequence of two peptides found in previous cross-linking studies reveals two stretches of primary structure that are presumably close in space to the reactive Cys-283 and hence close to the active site.     

Short chemoenzymatic synthesis of S-enantiomers of two systemic fungicides

Summary 2-Methyl-(4-tert-butyl)cinnamaldehyde (1) was reduced by Saccharomyces cerevisiae (baker's yeast) to S-3-(4-tert-butyl)-phenyl-2-propanol (4) in high chemical and very high optical yield (e.e. 99%). Chlorination of 4 to 5, and alkylation of the corresponding cyclic amines complete this short enantioselective synthesis of S-1-(l'-pyperidino)-2-methyl-3-(4tert-butyl)-phenyl-propane (6) and S-1-(1'-(3,5-cisdimethyl)morpholino)-2-methyl-3-(4-tert-butyl)-phenyl-propane (7), the S-enantiomers of fenpropidine and fenpropimorph, commercialy important systemic fungicides.