Two-dimensional crystals of enzyme-effector complexes: ribonucleotide reductase at 18-.ANG. resolution

Ribi, Hans O.; Reichard, Peter; Kornberg, Roger D.

doi:10.1021/bi00398a064

Cited by 59 publications

(23 citation statements)

References 18 publications

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“…Two-dimensional crystallization Two-dimensional crystallization of biological macromolecules using either charged (Darst et al, 1988(Darst et al, , 1989(Darst et al, , 1991Schultz et al, 1990a) or ligand exposing (Uzgiris and Kornberg, 1983;Ribi et al, 1987;Lebeau et al, 1990; Mosser et al, 1991) lipid layers has grown to become a powerful tool for electron microscopic studies of soluble proteins. These studies have shown that proteins are concentrated at the lipid -solvent interface upon interaction with the lipids and, in some cases, it has been shown that they are preferentially oriented (Schultz et al, 1990a (Darst et al, 1989(Darst et al, , 1990(Darst et al, , 1991.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

et al. 1993

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Section: Discussionmentioning

confidence: 99%

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

et al. 1993

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“…The length of the dimeric molecule is about 110 A and with a largest diameter of 75 A. We have compared our Rl structure with the object observed by image analysis of two-dimensional RI crystals [31]. Our suggestion is that the 110 A object they interpreted as an RI subunit more likely corresponds to one RI dimer.…”

Section: Resultsmentioning

confidence: 91%

Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli

Uhlin

Eklund

1993

FEBS Letters

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Crystals of Escherichia coli ribonucleotide reductase protein Rl have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2. Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 8, and c = 341 A. They contain 3 subunits per asymmetric unit and diffract to 2.5 8, resolution in synchrotron radiation. A multiple isomorphous replacement map at 5.5 A, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 A long. The dimer is thin in the middle around the molecular two-fold axis. The subunit is shaped like a bowl, probably with the active site in its center.Crystallization; Ribonucleotide reductase; Protein purification; Peptide complex; Crystal packing; Heavy atom derivative I~RDDUCTIONRibonucleotide reductase (RNR) is an essential enzyme for DNA synthesis. It catalyses the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides [l]. In Escherichiu coli and higher organisms RNR consists of two homodimeric unequal proteins Rl and R2 (for reviews see [24]). A catalytically active enzyme is formed by a one to one complex of the two proteins in the presence of Mg" and ATP. The carboxyl end of R2 contributes to a large extent to the binding between the two proteins, and peptides of the carboxyl end of R2 have been shown to inhibit formation of the active holoenzyme [5][6][7][8].Each subunit of the protein Rl has two different effector binding sites for allosteric regulation. One site regulates the overall activity with ATP and dATP as effecters while the other site regulates substrate specificity with deoxyribonucleoside triphosphates as effecters. Rl also contains redox active cysteines which become oxidized during catalytis. To regenerate active enzyme, this disulfide bridge has to be reduced. This can be done by at least two different systems, the glutaredoxin and thioredoxin systems 19).The Rl protein is about twice as large as the R2 protein in most species. In E. co/i, Rl has a molecular weight of 2 x 83 kDa where each peptide consists of 761 amino acid residues [lo]. The smaller R2 protein (2 x 43.4 kDa) contains a tyrosyl free radical close to a binuclear iron center [I 11. *Corresponding author.For several years we have worked on the structure dete~ination of ribonucleotide reductase from E. co&. The main difficulty of this work has been to obtain useful crystals for crystallographic studies. The R 1 protein has been crystallized in at least ten different crystal forms, and R2 in three orthorhombic crystal forms [12]. Most of the crystal forms were not suitable for highresolution crystallography. Crystals of the holoenzyme have been obtained, but they have large cell dimensions and are X-ray sensitive and thus not useful for structure determination. By extensive exploration of different crystallization conditions in combination with improved enzyme preparation we were able to obtain crystals of good quality...

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“…Ribi et al synthesized functionalized lipid 4 containing dATP linked via L--dioleoylphosphatidylethanolamine (DOPE) 1 to obtain 2-D crystals of the B1 subunit of ribonucleotide reductase, an ATP-binding protein [18].…”

Section: Atp-lipidsmentioning

confidence: 99%

“…Electron micrographs of negatively stained crystals diffracted to 18 Å (Fig. (2)) and revealed the structure of the B1 subunit to be a dimer, consisting of a pair of cylindrical objects, each of 105-109 Å length and 31-34 Å in diameter [18].…”

Section: Atp-lipidsmentioning

confidence: 99%

Synthetic Approaches to Functionalized Lipids for Protein Monolayer Crystallizations

Hussein¹,

Ross

Landsberg³

et al. 2009

COC

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The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localized concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2-D crystalline arrays that are amenable to structure determination by single particle analysis or 2-D electron crystallography. 2-D crystals grown by this technique can also be used to initiate the growth of 3-D crystals for X-ray diffraction analysis. Many derivatized lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalized head groups that chelate Ni 2+ or Cu 2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalized hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. This review focuses on synthetic approaches to functionalized lipids for protein monolayer crystallizations.

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Two-dimensional crystals of enzyme-effector complexes: ribonucleotide reductase at 18-.ANG. resolution

Cited by 59 publications

References 18 publications

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli

Synthetic Approaches to Functionalized Lipids for Protein Monolayer Crystallizations

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