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F Frolow

Publications and source records attributed to F Frolow.

At least 37 records · Page 2Linked to original sources
PubMed1994

Structure of a unique twofold symmetric haem-binding site.

Bacterioferritin of Escherichia coli, also known as cytochrome b1, is a hollow, nearly spherical shell made up of 24 identical protein subunits and 12 haems. We have solved this structure in a tetragonal crystal form at 2.9 A resolution. We find that each haem is bound in a pocket formed by the interface between a pair of symmetry-related subunits. The quasi-twofold axis of the haem is closely aligned with the local twofold axis relating these subunits. The axial ligands of the haem are sulphurs of two equivalent methionyl residues (Met 52) from the symmetry-related subunits. A cluster of four water molecules is trapped in the gap between the upper edge of the haem and two extended protein loops which close off the haem from the outer aqueous environment. This is the first structure of a bis-methionine ligated haem-binding site and the first case of a twofold symmetric haem-binding site.

Bacterial Proteins↗
PubMed1993

Location of haem in bacterioferritin of E. coli.

. A low-resolution partial structure of bacterioferritin was solved using a combination of molecular replacement and rigid-body refinement methods. Modification of bacterioferritin crystals by soaking in tetrachloroplatinate results in a phase transition from tetragonal symmetry (space group P4(2)2(1)2) to a pseudo-cubic one (approximate space group I432). Helical parts of human H ferritin structure stripped of side chains beyond the C(beta) atoms were used as the model. An electron-density map of the refined model revealed a region of extended density which by its shape and position in a pocket between helices was identified as haem. Inclusion of haem in the refinement showed that it can occupy only one of two symmetry-related sites near a twofold axis of the molecule.

Journal Article↗
PubMed1993

The conformation of new tetrahydropyrimidine derivatives in solution and in the crystal.

We have recently identified by NMR techniques two new tetrahydropyrimidine derivatives, 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], which accumulated in actinomycin D producing Streptomyces parvulus. Their relatively high intracellular concentrations during actinomycin D synthesis and identity of the time of onset of their synthesis with that of actinomycin D synthesis leads us to suggest that they may function in the self-defense mechanism of actinomycin-producing organisms. Here we present a combination of one-dimensional and two-dimensional 1H- and 13C-NMR studies in solution and X-ray crystallography of THP(A) and THP(B). Our results demonstrate identical conformations of THP(A) and of THP(B) in H2O solution and in the crystal. THP(A) and THP(B) form zwitterionic molecules with the half-chair conformation. Both techniques, NMR and X-ray crystallography, indicate that the carboxyl group of THP(B) is in an axial position and that the carboxyl and the hydroxyl groups of THP(A) are also in axial positions. The coplanar zigzag configurations (H4 C4 C5 C6 H6) of THP(A) and THP(B), deduced from NMR coupling measurements, were confirmed by X-ray diffraction studies. The crystal structure of THP(B) indicates that the hydrogen atoms of the methyl group are disordered, exhibiting two distinct conformations, whereas the methyl group hydrogens of THP(A) exhibit only one conformation. This finding is in accordance with the dynamic properties of THP(A) and THP(B) derived from carbon spin-lattice relaxation rate measurements. In spite of a different crystal packing and considerable differences in the dynamic properties (T1) in solution, the two molecules exhibit an almost identical conformation. The similar conformation of the two different crystals can be considered as evidence for a relatively high internal stability of the molecule against intermolecular forces.

Hydrogen Bonding↗
PubMed1993

Crystals of the phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 complexed with tRNA(Phe).

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from the extreme thermophile Thermus thermophilus HB8 has been crystallized with its cognate tRNA. Compared with the native crystals, the crystals of the complex are more stable to radiation damage and diffract to 3.0 A resolution. They are of space group P3(2)21, with a = b = 175 A, c = 142.1 A, gamma = 120 degrees, almost identical with the crystal parameters of the native synthetase.

Crystallization↗
PubMed1993

Crystal parameters of an alcohol dehydrogenase from the extreme thermophile Thermoanaerobium brockii.

A bacterial thermophilic alcohol dehydrogenase which is stable and active at 85 degrees C, has been crystallized by vapor diffusion from solutions of polyethylene glycol. A monoclinic crystal form diffracts to 2.8 A resolution and belongs to space group C2 with unit cell dimensions a = 139.0 A, b = 137.4 A, c = 80.9 A and beta = 93.23 degrees. The asymmetric unit contains four molecules which exhibit 222 point symmetry. A second crystal form is orthohombic, space group P2(1)2(1)2 with unit cell dimensions a = 168.0 A, b = 123.0 A, c = 80.0 A, and it diffracts to 3.2 A resolution.

Alcohol Dehydrogenase↗
PubMed1993

Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structures of bulge-containing DNA fragments.

The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 A resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A.A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 A resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.

Base Composition↗
PubMed1992

Interactions between acidic matrix macromolecules and calcium phosphate ester crystals: relevance to carbonate apatite formation in biomineralization.

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Earlier studies on the interactions between acidic macromolecules and carboxylate- and carbonate-containing crystals showed that the proteins recognize a specific stereochemical motif on the interacting plane. Here we show that a similar stereochemical motif is recognized by acidic mollusc shell macromolecules interacting with four different organic calcium phosphate-containing crystals. In addition, an acidic protein from vertebrate tooth dentin was also observed to recognize a similar structural motif in one of the crystals. The characteristic motif recognized is composed of rows of calcium ions and phosphates arranged in a plane defined by two free oxygens and a phosphorus atom emerging perpendicular to the affected face. These observations may have a direct bearing on the manner in which control over crystal growth is exerted on carbonate apatite crystals commonly found in vertebrate tissues.

Animals↗
PubMed1992

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.

Acetylcholinesterase↗
PubMed1991

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin.

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.

Alkylation↗
PubMed1991

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein.

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

Acetylcholine↗
PubMed1991

Gamma-chymotrypsin is a complex of alpha-chymotrypsin with its own autolysis products.

The determination of three separate gamma-chymotrypsin structures at different temperatures and resolutions confirmed the presence of electron density in the active site, which could be interpreted as an oligopeptide as had previously been suggested by Dixon and Matthews [(1989) Biochemistry 28, 7033-7038]. HPLC analyses of the enzyme before and after crystallization demonstrated the presence of a wide variety of oligopeptides in the redissolved crystal, most with COOH-terminal aromatic residues, as expected of the products of chymotrypsin cleavage, which appeared to arise from extensive autolysis of the enzyme under the crystallization conditions. The refined structures agree well with the conformation of both gamma-chymotrypsin and alpha-chymotrypsin. The electron density in the active site is thus interpreted as arising from a repertoire of autolysed oligopeptides produced concomitantly with crystallization. The COOH-terminal carbons of the polypeptide(s) display short contact distances (1.97, 2.47, and 2.13 A, respectively) to Ser195 O gamma in all three refined structures, but the electron density is not continuous between these two atoms in any of them. This suggests that some sequences are covalently bound as enzyme intermediates while others are noncovalently bound as enzyme-product complexes.

Amino Acid Sequence↗
PubMed1990

Crystallization and preliminary x-ray investigation of colicin E3 in complex with its immunity protein.

Crystals of the colicin E3-immunity protein complex have been grown from solutions of citrate at pH 5.6. The crystals are monoclinic, space group P2(1), with unit cell dimensions a = 67.71, b = 196.67, c = 85.58 A, and beta = 113.67 degrees. The crystals diffract to 3-A resolution and are stable in the x-ray beam for at least a day. Although the stoichiometry of the complex in solution is 1:1 there are two, three, or four such binary complex molecules in the asymmetric unit.

Bacterial Proteins↗
PubMed1990

The structure and hydration of the A-DNA fragment d(GGGTACCC) at room temperature and low temperature.

The DNA fragment d(GGGTACCC) was crystallized as an A-DNA duplex in the hexagonal space group P6(1). The structure was analyzed at room temperature and low temperature (100K) at a resolution of 2.5 A. The helical conformations at the two temperatures are similar but the low-temperature structure is more economically hydrated than the room-temperature one. The structure of d(GGGTACCC) is compared to those of d(GGGTGCCC) and d(GGGCGCCC). This series of molecules, which consists of a mismatched duplex and its two Watson-Crick analogues, exhibits three conformational variants of the A-form of DNA, which are correlated with the specific intermolecular interactions observed in the various crystals. The largest differences in local conformation are displayed by the stacking geometries of the central pyrimidine-purine and the flanking purine-pyrimidine sites in each of the three duplexes. Stacking energy calculations performed on the crystal structures show that the mismatched duplex is destabilized with respect to each of the error-free duplexes, in accordance with helix-coil transition measurements.

Base Sequence↗
PubMed1989

The conformation of the DNA double helix in the crystal is dependent on its environment.

Studies of the crystal structures of more than 30 synthetic DNA fragments have provided structural information about three basic forms of the double helix: A-, B- and Z-form DNA. These studies have demonstrated that the DNA double helix adopts a highly variable structure which is related to its base sequence. The extent to which such observed structures are influenced by the crystalline environment can be found by studying the same molecule in different crystalline forms. We have recently crystallized one particular oligomer in various crystal forms. Here we report the results of structural analyses of the different crystal structures and demonstrate that the DNA double helix can adopt a range of conformations in the crystalline state depending on hydration, molecular packing and temperature. These results have implications on our understanding of the influence of the environment on DNA structure, and on the modes of DNA recognition by proteins.

Base Composition↗
PubMed1989

Cryocrystallography of ribosomal particles.

Crystals suitable for X-ray study have been prepared from biochemically active ribosome particles or their complexes with tRNA and polypeptide chains. At ambient temperature the useful lifetime of these crystals under synchrotron irradiation is limited to a few minutes. However, upon cooling to cryogenic temperatures around 85 K, the original resolution limit (up to 4.5 A) can be recorded and radiation damage is virtually eliminated. Hence it has become possible to collect a complete data set from one single crystal. Crystals were cooled as rapidly as possible, either in a cold gas stream, or by immersion in liquid propane. Before cooling crystals were transferred either to an inert hydrocarbon environment, or to solutions similar to the crystallizing ones but with a higher viscosity. In several cases soaking in a cryosolvent was required. Crystallographic data were collected with intense synchrotron radiation. Full data sets have been measured for native and derivatized crystals of 50S ribosomal subunits from H. marismortui as well as from their complexes with tRNA and nascent polypeptide chains, from the wild type and a mutant of 50S subunits from B. stearothermophilus, and from crystals of native and derivatized 30S ribosomal subunits from T. thermophilus.

Bacteria↗
PubMed1988

Low-temperature study of the A-DNA fragment d(GGGCGCCC).

The structure of the A-type duplex d(GGGCGCCC) was determined from data measured at 115 K to 2.0 A resolution. The space group, P4(3)2(1)2, is the same as for the 293 K structure; cell dimensions are a = 42.74 (4), c = 24.57 (1) A; R = 0.21 for 1694 observed reflections. The conformation and hydration are similar at the two temperatures. The average displacement parameters (B) for bases, sugars and phosphates all decrease by about 9 A2 relative to those found at 293 K. The individual values of B1/2 are linearly related to the distance from the molecular center of mass.

Base Sequence↗