Supramolecular Self-Assembly of Escherichia coli Glutamine Synthetase: Effects of Pressure and Adenylylation State on Dodecamer Stacking

Atkins, William M.

doi:10.1021/bi00254a003

Cited by 8 publications

(10 citation statements)

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“…The number of moles of solvent ( n A ) and solute ( n B ) in the solution in osmotic equilibrium with pure solvent A. The slope of the ln λ i vs Π curve is defined by the relation1 where ∂V A ‡ is the change in hydration during the transition state. Estimated numbers of H 2 O molecules released in each respective transition state were calculated from the sucrose results among this set of osmolytes; about 71 molecules were released in the first subprocess and ca.…”

Section: Kineticsmentioning

confidence: 99%

“…An example of this approach was used to probe the requirement of the imidazole ring nitrogen atom to form a stable “guanine quartet” structure (Figure 1A). When key base nitrogens are removed from the Gs by replacing them with 7‐deazaguanine (dz 7 G**),1 G‐quartet formation can be selectively precluded. In contrast, one can use dimethyl sulfate to probe the residual exposure of GN7 sites after a quadruplex has folded and formed hydrogen bonds, revealing which Gs are bound and which are free 51.…”

Section: Introductionmentioning

confidence: 99%

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Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids

2000

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The dissociation and assembly of quadruplex DNA structures (and a few quadruplex RNAs) have been characterized at several levels of rigor, ranging from gross descriptions of factors that govern each process, to semiquantitative comparisons of the relative abilities of these factors to induce stabilization or destabilization, to quantitative studies of binding energies (thermodynamics), transformational rates (kinetics), and analysis of their transition‐state energies and mechanisms. This survey classifies these factors, describes the trends and focuses on their interdependencies. Quadruplex assembly is induced most efficiently by added K+ and elevating the strand concentration; however, Na+, NH4+, Sr2+, and Pb2+ are also very effective stabilizers. Quadruplex dissociation is typically accomplished by thermal denaturation, “melting”; however, when the quadruplex and monovalent cation concentrations are low enough, or the temperature is sufficiently high, several divalent cations, e.g., Ca2+, Co2+, Mn2+, Zn2+, Ni2+ and Mg2+ can induce dissociation. Stabilization also depends on the type of structure adopted by the strand (or strands) in question. Variants include intramolecular, two‐ and four‐stranded quadruplexes. Other important variables include strand sequence, the size of intervening loops and pH, especially when cytosines are present, base methylation, and the replacement of backbone phosphates with phosphorothioates. Competitive equilibria can also modulate the formation of quadruplex DNAs. For example, reactions leading to Watson–Crick (WC) duplex and hairpin DNAs, triplex DNAs, and even other types of quadruplexes can compete with quadruplex association reactions for strands. Others include nonprotein catalysts, small molecules such as aromatic dyes, metalloporphyrins, and carbohydrates (osmolytes). Other nucleic acid strands have been found to drive quadruplex formation. To help reinforce the implications of each piece of information, each functional conclusion drawn from each cited piece of thermodynamic or kinetic data has been summarized briefly in a standardized table entry. © 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 147–194, 2001

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Section: Kineticsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids

2000

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“…Assay for GS Stacking. The stacking reaction of wildtype and mutant GS proteins was monitored by 90°l ight scattering measurements and electron microscopy, as described in the accompanying papers (Yanchunas et al, 1994; Atkins, 1994). Samples contained 0.17 pM GS subunits in 50 mM Hepes, pH 7.0, 100 mM KC1, and 1 mM MnCl2 at 5 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Mutant proteins are identified by the standard system in which the single-letter amino acid abbreviation of the wild-type residue is followed by the amino acid number and the single-letter abbreviation for the amino acid substituted; e.g., H4A is the mutant in which histidine-4 is replaced with an alanine. 0006-2960/94/0433-14957$04.50/0 © 1994 American Chemical Society kinetics of this reaction (Yanchunas et al, 1994), and we have determined the kinetic parameters AH* and AV* for formation of the encounter complex en route to the "stacked" dodecameric assembly (Atkins, 1994). These parameters provide useful insights into the protein-solvent and proteinprotein interactions involved in GS stacking, but the mechanism for this macromolecular recognition remains unknown.…”

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confidence: 99%

Supramolecular Self-Assembly of Glutamine Synthetase: Mutagenesis of a Novel Intermolecular Metal Binding Site Required for Dodecamer Stacking

Dabrowski¹,

Yanchunas²,

Villafranca³

et al. 1994

Biochemistry

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Dodecameric glutamine synthetase (GS) from Escherichia coli assembles into highly ordered supramolecular protein tubes in the presence of several divalent metal ions. The molecular mechanism for this metal-induced self-assembly of the E. coli GS has been studied by molecular modeling and site-directed mutagenesis. The X-ray crystal structure of the nearly identical Salmonella typhimurium GS has been used to construct a model of the "stacked" complex between two dodecamers. A complementary fit, based on steric constraints, reveals a possible interaction between the N-terminal helices from adjacent dodecamers. The amino acid side chains of His and Met residues within the helices from each of the subunits of one face of a dodecamer lie within approximately 3.5 A of the analogous side chains in the subunits from the adjacent dodecamer in the stacked complex. His-4, Met-8, and His-12 from adjacent helices provide potential ligands for a binuclear metal binding site. Replacement of each of these surface residues with aliphatic amino acids has negligible effects on the enzymatic activity, the regulation of activity via adenylylation, and gross dodecameric structure. However, the rate and extent of metal ion-mediated self-assembly of GS tubules are reduced to < 2% of the wild-type protein in the single mutants H4A, H12L, and H12D. The M8L mutant demonstrates a 3-fold decrease in the bimolecular rate constant for stacking, but electron microscopy indicates that this mutant does form stacked tubes. The cysteine-containing mutants H4C, M8C, and H12C were also constructed.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…In addition, we have explored in vitro methods for removing the biologically imposed symmetry of the GS dodecamer, which allow for control over the length of GS tubes (12). Also, the solution conditions that influence this metal-dependent protein-protein interaction have been previously characterized for wild type and site-directed mutants of E. coli GS, in order to understand factors that control this process (8,13), and the thermodynamics of the reaction have been partially described (14). Together, these results have yielded GS variants with novel self-assembly properties and a useful structural model for the molecular mechanism of recognition between GS dodecamers.…”

Section: ؊1mentioning

confidence: 99%

Metal-dependent Self-assembly of Protein Tubes from Escherichia coli Glutamine Synthetase

Schurke¹,

Freeman²,

Dabrowski³

et al. 1999

Journal of Biological Chemistry

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Escherichia coli glutamine synthetase (GS) is a dodecameric assembly of identical subunits arranged as two back-to-back hexagonal rings. In the presence of divalent metal ions, the dodecamers "stack" along their six-fold axis of symmetry to yield elongated tubes. This self-assembly process provides a useful model for probing metal-dependent protein-protein interactions. However, no direct spectroscopic or structural data have confirmed the identity of the ligands to the shared metal ions in "stacked" GS. Here, 9-GHz Cu 2؉ EPR studies have been used to probe the ligand structure and stoichiometry of the metal binding sites. The wild type protein, with N-terminal sequence (His-4)-X 3 -(Met-8)-X 3 -(His-12), exhibits a classic Cu 2؉ -nitrogen spectrum, with g Ќ ‫؍‬ 2.06 G, g ʈ ‫؍‬ 2.24 G, and A ʈ ‫؍‬ 19.3 ؋ 10 ؊3 cm ؊1. No superhyperfine structure is observed. The H4C mutant affords a spectrum that is the combination of two spectra at all stages of saturation. One of the overlapping spectra is nearly identical to the spectrum of wild type, and is due to His ligation. The second spectrum observed yields g ʈ ‫؍‬ 2.28 and A ʈ ‫؍‬ 17.1 ؋ 10 ؊3 cm ؊1 . The linewidth and tensor values of the second component have been assigned to Cu 2؉ -S ligation. In contrast, the H12C mutant exhibits an EPR spectrum at low Cu 2؉ occupancy that is very similar to the second set of spectral features observed for H4C, and which is assigned to Cu 2؉ -S ligation. No Cu 2؉ -His ligation is apparent until the Cu 2؉ /N-terminal helices ratio is >1.0. At saturation, the g ‫؍‬ 2.00 -2.06 region of the spectrum is essentially a mirror image of the spectrum obtained with H4C, and is due to overlapping Cu 2؉ -N and Cu 2؉ -S EPR spectra. The M8L and M8C mutants were also studied, in order to probe the role of position 8 in the N-terminal helix. Spectral parameters of these mutants are nearly identical to each other and to the wild type spectrum at saturating Cu 2؉ , suggesting that Met-8 does not act as a direct metal ligand. Together, the results provide the first direct evidence for a binuclear metal ion site between each N-terminal helix pair at the GS-GS interface, with both His-4 and His-12 providing metal ligands.

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Supramolecular Self-Assembly of Escherichia coli Glutamine Synthetase: Effects of Pressure and Adenylylation State on Dodecamer Stacking

Cited by 8 publications

References 44 publications

Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids

Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids

Supramolecular Self-Assembly of Glutamine Synthetase: Mutagenesis of a Novel Intermolecular Metal Binding Site Required for Dodecamer Stacking

Metal-dependent Self-assembly of Protein Tubes from Escherichia coli Glutamine Synthetase

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