Elongated Conformation Research Articles

Structural and functional analysis of a bacterial cellulase by proteolysis

CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases.

Acid-induced dimerization of skeletal troponin C.

We have investigated pH-dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low-speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20 degrees C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20 degrees C, and was completely prevented at a rotor speed of 40,000 rpm and 4 degrees C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met-25 and Cys-98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH-dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The proton-induced increase in the energy transfer distance is related to the acid-induced dimerization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational flexibility in the molecular structure of rotenone, a naturally occurring insecticide

The crystal and molecular structure of rotenone, a naturally occurring insecticide with mitochondrial and mitotic spindle inhibitory properties, was determined by direct methods. The crystals were orthorhombic, space group, P2 I2 I2 I with two molecules in the asymmetric unit; a = 8.413 (1) A ̊ , b = 19.840(1), c = 23.581(1), V = 3936 A ̊ 3 , Z = 8. The structure was refined by least-squares methods to a final R = 0.067. The two molecules in the asymmetric unit have different conformations about the junction between the nonaromatic rings B and C. Ring B is in a sofa conformation in both molecules, with a slight distortion toward a half-chair in I, but with C8 and C8′ on opposite sides of the planar part of the rings. This difference in conformation results in I having an extended (linear) shape while II is V-shaped. The more elongated conformation of the molecule (I) has not been reported in previous studies. Ring C also has opposite conformations in the two molecules. The angle between the planes formed by rings A and D in molecule I is 64.3°, while in molecule II it is 88.3°. Molecular mechanics techniques were used to determine the energy of the two conformations. These calculations, at room temperature, predict molecule II to be the more stable conformer. The highly flexible site in the B/C ring junction is also chemically very reactive. This flexibility and reactivity are further discussed in terms of rotenone's inhibitory activities.

PH-dependent structural transition in rabbit skeletal troponin C.

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans of other products of smooth muscle cell metabolism.

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells.

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH3, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH3 on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with 125I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available 125I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH3 cultured in the serum-supplemented and the defined medium.

Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response.

Cultured rat pituitary tumor cells, GH3/D6, which synthesize both growth hormone and prolactin, have cell-surface epidermal growth factor (EGF) receptor sites (34,000 per cell) that bind 125I-labeled EGF with a high affinity (Kd approximately 1 nM). Prolonged treatment of the cells with EGF did not stimulate cell division but did inhibit thyroid hormone-stimulated cell growth. In addition, EGF altered the morphology of the cells from a rounded to an elongated conformation. EGF also induced a perturbation of chromatin structure in GH3 cell nuclei that was detected by an increase (40%) in the number of rifampicin-resistant initiation sites for bacterial RNA polymerase. This was accompanied by an increased synthesis of prolactin and an inhibition of synthesis of growth hormone. In the presence of EGF, the synthesis of growth hormone was no longer inducible by thyroid hormone, but it remained responsive to glucocorticoids. The results demonstrate that EGF can elicit major effects on the cellular phenotype and expression of specific genes in the absence of a proliferative response. This suggests that EGF can also regulate differentiated cellular functions.

Conformation of n-alkane molecules in the melt and in cyclohexane solution studied by small-angle neutron scattering

Small-angle neutron scattering experiments on hexadecane and hexatriacontane molecules in various states have been performed. The molecules in the melt and in cyclohexane solution show the same scattering behavior. The measured radii of gyration are in good agreement with those calculated by Flory on the basis of the rotational isomeric state theory. There is no evidence for any intermolecular interaction that favors elongated chain conformations. One must rather come to the conclusion that n-alkane molecules assume their unperturbed dimensions in the molten state.

Conformational Studies on C-Phycocyanin from Spirulina platensis

Abstract The chromophore-protein interactions of C-phycocyanin (C-PC) from Spirulina platensis have been studied by following the partial and complete denaturation with UV-Vis spectroscopy. From comparison with published MO calculations, an elongated conformation of the chromophore is suggested for native C-PC, a cyclic one for denatured C-PC. By means of partial denaturation, a step­ wise unfolding of the protein has been demonstrated. The presence of at least two sets of spectro­ scopically different diromophores is suggested from the partial denaturation and low temperature experiments.

Ribosomal proteins S5, S11, S13 and S19 localized by ekectron microscopy of antibody-labeled subunits

Specific antibodies prepared against purified Escherichia coli small subunit proteins were used to map protein locations by electron microscopy of bound antibody in negatively contrasted small subunit preparations. By identifying and interpreting characteristic views as projections of a single structure, the protein distributions on the surface of the small subunit have been mapped in three dimensions. Antibodies against ribosomal proteins S5, S11 and S13 bind to single regions of the ribosome surface. In contrast, anti-S19 immunoglobulin G binds to two separated regions of the ribosome surface, suggesting that S19 has an elongated conformation in situ. Proteins S13 and S19 are located on the smaller portion of elongated profiles, while S5 and S11 are located on the larger portion of the subunit profile, near the partition traversing the subunit image. The locations of exposed regions of S11, S13 and S19, in combination with the crosslinking results of others, suggest a binding site for initiation factor IF3 on a region of the 30 S surface, comprising the platform, the cleft, part of the partition, and part of the upper “one third” of the subunit.

Localization of Escherichia coli ribosomal protein S4 on the surface of the 30S ribosomal subunit by immuno electron microscopy

The location of the ribosomal protein S4 on the surface of the 30S subunit of E. coli ribosomes was determined by immuno electron microscopy. Immunoglobulins from six separate S4-specific antisera were investigated. In accordance with earlier findings protein S4 was shown to have an elongated conformation within the antisera. Protein S4 has therefore definitely an extended or fibrous shape in the intact ribosome (Lake et al., 1974; Stöffler and Tischendorf, 1975; Tischendorf et al., 1975). S4 specific antibodies bind at four distinct sites of 30S ribosomal subunits, designated A, B, C and D. Two antibody binding sites (A and B) are located on the "head", they were shown to be separated by 70-85 A. The distance between these sites and the two sites C and D on the "body" of the subunit amounts to at least 90-125 A; hence protein S4 should be extended to a total length of approximately 160-200 A. At least three of the four S4-specific antibody binding sites were observed with antibodies from each of the six investigated. These sites were observed independently of whether isolated S4 protein, an S4-16S rRNA complex or 30S ribosomes were used as the antigen. They could also be visualized with monovalent antibody fragments (Fab). S4-specific antibodies enriched by affinity chromatography bound at identical sites which conclusively ensures that antibody binding at these sites is specific for protein S4 and is not due to the presence of contaminating antibodies in the intact ribosome. The implications of these results with respect of the correlation of each of the sites to their respective antigenically active fragments within protein S4 are discussed.

Conformational difference between 8-bromo-2′-O-triisopropylbenzenesulfonyl-adenosine and its 3′-isomer determined by x-ray method

The crystal structures of 8-bromo-2′-O-triisopropylbenzenesulfonyl-adenosine and its 3′-isomer have been determined by X-ray diffraction method. In the former crystal, an intra-molecular stacked conformation between adenine- and benzene-ring was found and there exist two modes of dimer formation: an inter-molecular stacked dimer and a hydrogen-bonded one. On the other hand, the 3′-isomer, 8-bromo-3′-O-triisopropylbenzenesulfonyladenosine, has a similar elongated non-stacked conformation as puromycin.

Molecular properties of multiple forms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii

1. 1. Using methods of gel filtration, viscosity, sedimentation velocity and sedimentation equilibrium, the molecular weights of the two isoenzymes of phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating), EC 4.1.1.31) from Chlamydomonas reinhardtii have been determined. Values ranged from 1.00·10 5–1.18·10 5 for the A conformer and 2.79·10 5–2.90·10 5 for the B conformer. 2. 2. On the basis of determination of the shape function constant, frictional coefficient and axial ratio, the A conformer appeared to possess a compact spherical type of conformation compared to a more elongated conformation for the B isoenzyme.

Determination of the stokes radius of native and succinylated glutamate dehydrogenase on sepharose 4B

The Strokes radius of native and of succinylated beef liver glutamate dehydrogenase was measured by gel filtration on Sepharose 4B. Methods for the determination of the Strokes radius of a protein on a gel were compared. The succinylated enzyme was found to lose all glutamate dehydrogenase activity and has a very elongated conformation.

FORMACIÓN DE RECURSOS Y DESARROLLO DE CAPACIDADES DE LOS MÚSICOS. EL CASO DE LA ESCUELA SUPERIOR DE MÚSICA SAGRADA DE LA ARQUIDIÓCESIS DE GUADALAJARA

The cauliflower mosaic virus (CaMV) 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600-nt leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and packaging. We have recently documented that the 35 S RNA leader adopts an elongated hairpin conformation and additional higher-order structures, a long-range pseudoknot and a dimer. Alternative structures might coexist, probably fulfilling specialized functions. In this paper, we analyze the biological significance of the elongated hairpin structure. We have introduced a spectrum of large deletions and small substitutions in the 35 S RNA leader and characterized their impact on the structure by temperature gradient gel electrophoresis. This analysis showed that the elongated hairpin consists of three sections of different stability (stem section I, II, and III). The overall secondary structure is relatively stable in the range of 10-32 degrees C. It melts between 32 and 38 degrees C in a manner indicating that the most stable base pairing occurs at the base of the elongated hairpin (stem section I). Mutations that destabilize stem section I decrease both the melting temperature of the leader and the expression in vitro and in vivo of the downstream CAT-reporter gene. Compensatory mutations restoring the stable elongated hairpin upregulate the translation efficiency. Our results demonstrate that the regulation of translation of the CaMV 35 S RNA is mediated by a stable hairpin in the leader.

Stereochemistry of nucleic acids and their constituents. IX. The conformation of the antibiotic puromycin dihydrochloride pentahydrate.

The elucidation of puromycin structure represents the first case where both a nucleic acid and a protein fragment are found together in a crystal. The molecule displays an elongated conformation with the nucleoside and the p-methoxy-L-phenylalanine in the preferred conformation. The purine and the tyrosine rings form alternating stacks with interplanar spacings of 3.4 A. The inhibitory effect of this antiobiotic may be due, in part, to its unique conformation and the ability of the amide proton on N(3') to hydrogen bond to the active site of the ribosomal enzyme, peptide synthetase.

Physical and chemical studies on glycoproteins: IV. The influence of sialic acid on the conformation of fetuin

The influence of sialic acid on the conformation of fetuin was investigated by comparing desialized fetuin with untreated fetuin using four different physical chemical techniques. The strong contribution of sialic acid to the acidic properties of fetuin was shown by the rise in the isoionic point from 4.23 to 5.57 for desialized fetuin. Comparison of the titration curves for both the desialized and untreated fetuin revealed that the number of titratable groups in the acidic, neutral, and basic regions agreed with the number expected from the amino acid analysis. The main difference between the two curves was the presence of additional titratable groups in the titration curve of fetuin in the region below pH 4 where the sialic acid carboxyl groups titrate. No evidence was found for the presence of free amino groups of the hexosamine residues, indicating that the amino sugar exists in fetuin as the acetylated derivative. There was no significant difference between the experimental electrostatic interaction factor w, 0.031, whose calculation is based on the carboxyl groups of desialized fetuin, and the experimental value, 0.029. The results from viscosity studies show that both proteins are fairly compact molecules having a conformation approximated by a prolate ellipsoid of revolution with an axial ratio of 5:1. There was little difference in the intrinsic viscosity of fetuin (5.5 ml/g) and desialized fetuin (5.2 ml/g). These data indicate that no appreciable conformational changes take place on removal of the sialic acid from fetuin. The results from optical rotatory dispersion and circular dichroism studies were inconclusive; at most only a small percentage of secondary structure was indicated. It was concluded that fetuin has a slightly elongated but compact molecular conformation possibly having some secondary structure; removal of the sialic acid does not alter this structure appreciably.