Biomedical subjects

D Tsernoglou

Publications and source records attributed to D Tsernoglou.

At least 37 records · Page 2Linked to original sources

PubMed1994

Crystallization of an enzyme-inhibitor complex: proteinase K with its protein inhibitor, PK13.

Crystals of a complex of proteinase K (molecular mass, 28,790 Da) with its naturally occurring protein inhibitor PK13 (19,641 Da), have been prepared by a microdialysis technique and modified by hanging drop vapor diffusion against 25% ammonium sulfate in 50 mM Tris-HCl, pH 7.8. The crystals are long prisms with diamond-shaped cross sections of 0.2 x 0.4 x 1.5 mm3 and they diffract X-rays to a resolution of 2.5 A. They belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 64.1 A, b = 66.8 A, and c = 133.8 A. Assuming one whole complex in the asymmetric unit, one obtains VM = 2.95 A3/Da and the solvent content, Vsolv = 58.3%.

Chromatography, Gel↗

PubMed1994

Restored heptad pattern continuity does not alter the folding of a four-alpha-helix bundle.

The sequences of alpha-helical coiled-coils and bundles are characterized by a specific pattern of hydrophobic and hydrophilic residues which is repeated every seven residues. Highly conserved breaks in this pattern frequently occur in segments of otherwise continuous heptad substructures. The hairpin bend of the ROP protein coincides with such a break and provides a model system for the study of the structural effects induced by heptad discontinuities. The structure of a ROP mutant which re-establishes a continuous heptad pattern, shows insignificant changes relative to the wild-type protein, as is also reflected in its conformational stability, spectroscopic properties and unfolding behaviour. Thus, formation of alpha-alpha-hairpin bends may occur both in the presence and absence of heptad breaks.

Amino Acid Sequence↗

PubMed1993

Correlation between protein stability and crystal properties of designed ROP variants.

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-alpha-helical bundles, have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolution X-ray diffraction studies. For all mutants crystal size, sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry, in a manner which is not yet understood in detail.

Bacterial Proteins↗

PubMed1993

The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme.

Amino Acid Sequence↗

PubMed1992

Refined structure of the pore-forming domain of colicin A at 2.4 A resolution.

The E1 subgroup (E1, A, B, IA, IB, K and N) of anti-bacterial toxins called colicins is known to form voltage-dependent channels in lipid bilayers. The crystal structure of the pore-forming domain of colicin A from Escherichia coli has been refined to the diffraction limit of the crystals at 2.4 A resolution by means of molecular dynamics and restrained least-squares methods to a conventional R-factor of 0.18 for all data between 6.0 and 2.4 A resolution. The polypeptide chain of 204 amino acid residues consists of ten alpha-helices organized in a three-layer structure. The helices range in length from 9 to 23 residues with an average length of 125 residues. The packing arrangement of the helices has been analysed; the packing is different from that observed in four-helix bundle proteins. The sites of 83 water molecules have been located and refined. Analysis of the structure provides insights into the mechanism of formation of a voltage-gated channel by the protein. Although it is proposed that substantial tertiary structural changes occur during membrane insertion, the secondary structural elements remain conserved. This idea has been proposed recently for a number of other protein-membrane events and thus may have more general applicability.

Amino Acid Sequence↗

PubMed1991

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.

Bacterial Outer Membrane Proteins↗

PubMed1991

Role of glycine-82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein.

Ras proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins.

Amino Acid Sequence↗

PubMed1991

A common channel-forming motif in evolutionarily distant porins.

Four new crystal packings of Escherichia coli porins are presented (phosphoporin, maltoporin, and two crystal forms of matrix porin). These were determined by molecular replacement methods using a polyalanine trial model acquired from the refined coordinates of porin from Rhodobacter capsulatus. The successful molecular replacement shows that the dominant motif found in R. capsulatus porin (a 16-stranded antiparallel beta-barrel) also applies to the E. coli porins, despite the lack of significant amino acid sequence homology. A 30 degrees-40 degrees tilt of the beta-strands with respect to the membrane normal was derived from the intensity distributions in the X-ray diffraction patterns for each porin studied, stressing their similarity. In view of the evolutionary distance between enteric and photosynthetic bacteria, the antiparallel beta-barrel may have significance as a basic structural motif for the formation of bacterial membrane channel structures.

Bacterial Outer Membrane Proteins↗

PubMed1990

Crystallization of a proform of aerolysin, a hole-forming toxin from Aeromonas hydrophila.

Crystals of proaerolysin, the precursor of the hole-forming toxin from Aeromonas hydrophila, have been obtained. The mature form of this protein binds to a receptor on mammalian cells, aggregates and forms 30 A holes in the membrane. The crystals are tetragonal, space group P4(3)2(1)2, a = b = 104.00 A, c = 222.0 A. They contain a dimer in the asymmetric unit and diffract to a resolution of 2.6 A.

Aeromonas↗

PubMed1990

Colicins: prokaryotic killer-pores.

Colicins are plasmid-encoded protein antibiotics which kill bacteria closely related to the producing strain (generally Escherichia coli). The study of the function of colicins has revealed many features which reflect common targeting and translocation mechanisms with bacteriophages and toxins. Like many toxins, colicins are composed of structural domains specialized in one of the different steps of the activity, targeting, translocation and killing. The major group comprises those colicins which permeabilize the cytoplasmic membrane, thereby destroying the cell's membrane potential. These colicins form well-defined voltage-gated ion channels in artificial membranes. The scope of this review is to describe some of the more recent findings concerning the structure and mode of action of pore-forming colicins with a special attention to models of membrane insertion and pore structure based on the recently determined three-dimensional structure of the pore-forming domain of colicin A.

Amino Acid Sequence↗

PubMed1990

Insights into membrane insertion based on studies of colicins.

The recently determined three-dimensional structure of the pore-forming domain of colicin A has led to a hypothetical model for membrane insertion and channel formation. Certain features of this model have implications for understanding the mechanism of membrane insertion by other toxins and may have a broader relevance to protein transport in general.

Biological Transport↗

PubMed1989

Application of molecular dynamics in the crystallographic refinement of colicin A.

Crystallographic refinement based on molecular dynamics (MD) has been applied to a 2.5 A resolution X-ray structure of the pore-forming fragment of colicin A. The crystallographic R factor was reduced from 48 to 23% with a concomitant improvement in stereochemical parameters. The method considerably speeded up the refinement process but was associated with some pitfalls. In particular, some badly fitted segments of the structure required manual rebuilding, even after MD refinement and some problems with weighting schemes were encountered. Analysis of the effects of the refinement and ideas for improvements are presented.

Colicins↗

PubMed1989

Crystallization of p68 on lipid monolayers and as three-dimensional single crystals.

Two-dimensional crystals of p68, a Ca2+ -binding protein that has homology with members of the lipocortin/calpactin family, were obtained by interaction with a phospholipid monolayer. By measuring surface pressure at constant surface area, p68 was found to interact in a Ca2+ -dependent manner specifically with phosphatidylethanolamine, less so with phosphatidylserine and not at all with phosphatidylcholine. With dimyristoyl-phosphatidylethanolamine, two-dimensional crystalline arrays were formed. Image analysis of electron micrographs of these crystals, which diffracted to about 50 A, revealed p3 symmetry with a unit cell of about 178 A by 178 A; the protein densities showed a two-domain structure giving a cylindrical molecule of about 100 A by 35 A diameter packed as trimers. Three-dimensional microcrystals obtained without lipid or Ca2+ were suitable for electron microscopy and gave a tetragonal unit cell of about 256 A by 68 A. The implications of these observations on the structure and lipid specificity of p68 binding are discussed.

Annexin A6↗

PubMed1989

Structure of the membrane-pore-forming fragment of colicin A.

Colicins are antibiotic proteins produced by and active against sensitive Escherichia coli and closely related bacteria. They can adsorb to specific receptors located at the external surface of the outer membrane of sensitive cells, and are then translocated to their specific targets within these cells. The largest group of colicins comprises those which can form voltage-dependent channels in membranes, thereby destroying the cell's energy potential. Colicin molecules are organized in structural domains, each domain carrying one function associated with the toxin's lethal activity. The pore-forming activity seems to be located at the carboxyl terminus. A thermolytic fragment comprising amino acids 389-592 from colicin A has pore-forming properties very similar to those of the entire molecule. This fragment is soluble in aqueous medium and spontaneously inserts into lipid bilayers. We have determined the structure of the pore-forming fragment of colicin A by X-ray crystallography and refinement at 2.5 A resolution. The protein consists of ten alpha-helices organized in a three-layer structure. Two of the helices are completely buried within the structure and form a hydrophobic hairpin loop similar to that proposed for signal sequences which function in translocation. We present a model for insertion of the protein into lipid bilayers the features of which may be applicable in other biological systems involving protein insertion or translocation across membranes.

Amino Acid Sequence↗

PubMed1989

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6.

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.

Alcaligenes↗

PubMed1989

Genetic and structural analysis of the ColE1 Rop (Rom) protein.

Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14.

Amino Acid Sequence↗

PubMed1989

Crystallographic phases through genetic engineering: experiences with colicin A.

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. The structure of the pore-forming fragment of colicin A has been solved to 2.5 A resolution using the techniques of X-ray crystallography and genetic engineering. Site-directed mutagenesis was used to construct a number of cysteine-containing mutant proteins, one of which yielded an excellent mercurial derivative. Our experiences suggest strategies for obtaining useful heavy-atom derivatives for protein crystallography using genetic engineering techniques.

Amino Acid Sequence↗

PubMed1987

Structure of the ColE1 rop protein at 1.7 A resolution.

Structural details of the Rop protein from plasmid ColE1 are presented, with a description of the X-ray crystal structure determination and refinement at a nominal resolution of 1.7 A. The 63 amino acid protein is a dimer. Each monomer consists almost entirely of two alpha helices, the whole molecule forming a highly regular four-alpha-helix bundle. This may be approximated by a four-stranded rope with a radius of 7.0 A, a left-handed helical twist and a pitch of 172.5 A. The packing constraints for this novel type of coiled-coil structure are given. The protein acts in the control of plasmid replication via regulation of an RNA-RNA interaction in a manner not yet understood in atomic detail.

Amino Acid Sequence↗