Tsunehiro Takano

High-salt d(CpGpCpG), a left-handed Z′ DNA double helix

The DNA tetramer d(CpGpCpG) or CGCG crystallizes from high-salt solution as a left-handed double helix, the Z′ helix. Its structure differs from that of the other known left-handed helix, Z-DNA, by a Cl′-exo sugar pucker at deoxyguanosines rather than C3′-endo, and these represent two alternative solutions to the same steric constraint arising from the syn glycosyl bond orientation. The apparent molecular basis for the Z to Z′ transition in going from intermediate to high salt is substitution of a bound anion for water at guanine amino groups, and consequent charge repulsion of anions and backbone phosphates.

Conformation change of cytochrome c: I. Ferrocytochrome c structure refined at 1·5 Å resolution☆

Abstract Tuna ferrocytochrome c has been crystallographically refined at a resolution of 1.5 A using the Diamond real-space method followed by Jack-Levitt restrained energy and reciprocal space refinement, monitoring progress continuously with superimposed Fourier and difference Fourier maps: The final R factor for cytochrome plus 53 solvent molecules, using 13,840 reflections with intensities greater than 2 σ, is 17·3%. The overall structure remains as described earlier (Takano et al., 1977), but structural details have been clarified to the point where meaningful comparison can be made with the oxidized molecule (following paper). Main and side-chain flexibility as judged by isotropic temperature parameters correlate with position in the molecule, with greatest flexibility at external chain loops. The haem group is held tightly in place by its attachments and neighbours, and is deformed slightly into a saddle shape. The iron does not deviate significantly from the best mean plane of the haem, and bond lengths to ligands are as expected from model compounds. A water molecule buried in the haem crevice is bonded to Asn52, Tyr67 and Thr78, the latter two being bonded also to Met80 and the outer haem propionate. It is proposed that this buried water molecule is involved in the reduction of ferricytochrome c by chromous ion, and the reactions of Tyr67 with KI3 and tetranitromethane. Two other buried water molecules occur beneath the 20s loop at the right, and within the 40s loop at the bottom. Reasonable if tentative functional assignments can be made for all 24 of the evolutionarily invariant residues in the cytochrome molecule.

Conformation change of cytochrome c. II. Ferricytochrome c refinement at 1.8 A and comparison with the ferrocytochrome structure.

Tuna ferricytochrome c has been crystallographically refined at a resolution of 1·8 A using Diamond real-space methods followed by Jack Levitt restrained energy and reciprocal space refinement, with Fourier and difference Fourier map monitoring. The final R factor for two independent cytochromes plus 49 solvent molecules, using 16,831 reflections to 1.8 A is 20·8%. The structure is essentially the same as described at 2.0 A (Swanson et al., 1977), but the increased accuracy now permits detailed comparison with the reduced cytochrome c molecule (preceding paper). Twelve water molecules are found in identical positions in both oxidized molecules and also in the reduced. Three of these are buried: one between Asn52, Tyr67 and Thr78 to the lower left of the haem, a second inside the 20s loop, and a third below the buried haem propionate. The first of these three buried water molecules is involved in a concerted shift of side-chains and even main-chain at the bottom of the molecule, that accompanies the change in redox state of the haem. The shift of this water molecule and a slight outward movement of the haem both give the haem a more hydrophilic environment in ferricytochrome c. This same buried water molecule may be involved in the alkaline conformation transition that ferricytochrome c undergoes at pH 9·4, assisting in the replacement of Met80 by a different low-spin ligand. The absence of such transition in ferrocytochrome c until pH 12 is reached may be ascribed to the shift in water position observed in the reduced state. The water molecule buried in the 20s loop, in turn, may be involved in the acid transformation that ferricytochrome c undergoes at pH 2·5. The conformations observed by X-rays for both ferricytochrome c and ferrocytochrome c appear to be post-transfer states, i.e. states that are most compatible with the particular redox state of the haem, rather than being poised to give up or receive electrons. These conformations have been interpreted as being effects of changes in haem redox state rather than causes, but if binding to another macromolecule were to induce either conformation, then transfer of an electron might thereby be facilitated.

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

Abstract The molecular structures of ferri- and ferrocytochrome c 551 from Pseudomonas aeruginosa have been refined at a resolution of 1.6 A, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F obs , and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state. Pseudomonas cytochrome c 551 differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c . As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c 551 than in cytochrome c .

Crystal data, molecular dimensions and molecular symmetry in cytochrome oxidase from Pseudomonas aeruginosa

Pseudomonas aeruginosa cytochrome oxidase (nitrite reductase, cytochrome cd) has been crystallized in space group P21212 with cell dimensions a = 122.8 A, b = 87.2 A, c = 73.4 A. Density measurements suggest that the asymmetric unit contains one 63,000 molecular weight subunit of the dimeric molecule. Crystal data agree well with electron microscopy of single molecules. The X-ray pattern extends beyond 2.5 A resolution, and structure analysis is in progress.

Large-scale purification of two forms of active lac operator from plasmids

A practical procedure is described for obtaining milligram quantities of a small (29 nucleotide) Eco RI restriction fragment of DNA containing the Escherichia coli lac operator. A yield of 10--15 mg of operator is obtained from 1 kg of wet cell paste. The resultant operator is shown to be homogeneous and competitively active in filter assays. Two separable but interconvertible forms of lac operator exist in solution, probably linear duplex and hairpin isomers. Only the presumed linear form is active in binding lac repressor by competition assay, but the two isomers are interconvertible by heating to 80 degrees C. The methods described here should be generally applicable for purifying other restriction fragments from plasmids.

FERRICYTOCHROME c: II. CHAIN FLEXIBILITY AND A POSSIBLE REDUCTION MECHANISM †

ABSTRACT The recent high resolution crystal structure analysis of horse and bonito ferricytochromes c (Dickerson et al., J. Biol. Chem. , 1971, in press) has shown the polypeptide chain path and the positions of all side chains. Evidence from chemical modification of side chains—iodination and nitration of tyrosines, carboxymethylation of methionines, and oxidation of tryptophans—interpreted in the light of the molecular structure, suggests that the left side of the molecule is flexible to a certain extent, and is essential for the reduceability of ferricytochrome c. A free radical mechanism for the reduction of ferricytochrome c , first proposed by M. E. Winfield in 1965 ( J. Mol. Biol. 12, 600), is presented and is shown to be compatible with present structural and chemical information about the left side of the molecule. This mechanism provides different pathways for the electron in reduction and oxidation, and obviates the necessity for the molecule to rotate upon interacting with its reductase and oxidase.