Seved Lövgren

Structure of Satellite tobacco necrosis virus at 3.0 Å resolution

Abstract The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 A resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.

Crystallization of a non-muscle actin

Eukaryotic cells contain a protein which specifically inhibits DNAsae I. This inhibitor protein is closely related to muscle actin. As shown here the inhibitor purified from calf spleen consists of two polypeptides: one which closely resembles muscle actin and a second (15,000 to 20,000 in molecular weight) whose combination with actin has not been recognized before. Crystals of the complex were obtained in ammonium sulphate. They belong to the orthorhombic space group P212121, have unit cell dimensions of a = 187.4 A, b = 72.33 A and c = 38.19 A, and have one molecule of the spleen actin associated with one molecule of the low molecular weight protein per asymmetric unit.

Urea-PETT compounds as a new class of HIV-1 reverse transcriptase inhibitors. 3. Synthesis and further structure-activity relationship studies of PETT analogues

The further development of allosteric HIV-1 RT inhibitors in the urea analogue series of PETT (phenylethylthiazolylthiourea) derivatives is described here. The series includes derivatives with an ethyl linker (1-5) and racemic (6-16) and enantiomeric (17-20) cis-cyclopropane compounds. The antiviral activity was determined both at the RT level and in cell culture on both wild-type and mutant forms of HIV-1. Most compounds have anti-HIV-1 activity on the wt in the nanomolar range. Resistant HIV-1 was selected in vitro for some of the compounds, and the time for resistant HIV-1 to develop was longer for urea-PETT compounds than it was for reference compounds. Preliminary pharmacokinetics in rats showed that compound 18 is orally bioavailable and penetrates well into the brain. The three-dimensional structure of complexes between HIV-1 RT and two enantiomeric compounds (17 and 18) have been determined. The structures show similar binding in the NNI binding pocket. The propionylphenyl moieties of both inhibitors show perfect stacking to tyrosine residues 181 and 188. The cyclopropyl moiety of the (+)-enantiomer 18 exhibits optimal packing distances for the interactions with leucine residue 100 and valine residue 179.

Structure of human carbonic anhydrase B: I. Crystallization and heavy atom modifications

Abstract Human carbonic anhydrase B has been crystallized from 2.3 m -ammonium sulphate solution at pH 8.7. A method for reproducible crystallization is presented. The crystals are suitable for high-resolution X-ray diffraction studies. They belong to the orthorhombic space group P 212121 with cell dimensions a = 81.5 A , b = 73.6 A , c = 37.1 A . The position of the essential zinc ion has been established from two projections. The zinc ion has been replaced by mercury to form one of the heavy atom derivatives.

Structure and function of carbonic anhydrase: comparative studies of sulphonamide binding to human erythrocyte carbonic anhydrases B and C

Soon after the discovery of carbonic anhydrase from bovine erythrocytes (E.C.4.2.1.1), which catalyses the reaction by Meldrum and Roughton (1932) it was shown by Mann and Keilin (1940) that certain sulphonamides were specific and potent inhibitors of this enzyme. It has been well documented (see Maren, 1967, for an extensive review on inhibition and physiology, and Lindskog et al. 1971, and Coleman, 1971, for general reviews on carbonic anhydrases) that aromatic and heterocyclic sulphonamides with unsubstituted R—SO2 NH2 groups constitute a class of powerful inhibitors, while aliphatic sulphonamides are poor inhibitors of the carbonic anhydrases.

Functional Aspects of the Three-Dimensional Structure of the Active Site of Carbonic Anhydrase

Carbonic anhydrase, which is found in large quantities in red cells is an extremely powerful catalyst of the hydration of carbon dioxide (Khalifah, 1971). The dehydration of bicarbonate is also catalysed at an appreciable rate (Christiansen and Magid, 1970). The enzyme is able to hydrate certain aldehydes (Pocker and Meany, 1965) and hydrolyse a number of esters (Thorslund and Lindskog, 1967).

Increased yield of homogeneous HIV-1 reverse transcriptase (p66/p51) using a slow purification approach

A chromatographic procedure to purify recombinant reverse transcriptase (RT) from human immunodeficiency virus-1 is reported. A bacterial system which expressed large amounts of p66 RT polypeptide was used. The purification scheme was optimized for high-yield production of homogeneous p66/p51 RT using a combination of chromatographic matrices in the following order: Q-Sepharose, heparin-Sepharose, phenyl-Sepharose, S-Sepharose, Poly(A)-Sepharose and Q-Sepharose. The p66 polypeptide remained intact after the first chromatographic step on Q-Sepharose, where it was recovered in the non-adsorbed fraction. A high yield of p66/p51 RT was obtained when the time from application to elution of heparin-Sepharose in the second chromatographic step was prolonged. Phenyl-Sepharose was used in the next chromatographic step to separate the heterodimeric forms of RT from p66 RT on the basis of hydrophobicity. The chromatography on S-Sepharose resolved the major heterodimeric form, p66/p51, from other heterodimeric variants. Further purification was done by affinity chromatography on Poly(A)-Sepharose followed by anion-exchange chromatography on Q-Sepharose. Amounts of 25-35 mg of the pure heterodimer p66/p51 RT were recovered from 50 g of bacterial cells.

Purification, characterization and crystallization of recombinant HIV-1- reverse transcriptase

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).

Crystallization and crystallographic data of notexin: A neurotoxic basic phospholipase A from the venom of Australian tiger snake Notechis scutatus scutatus

Abstract Crystals of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake) have been grown in 1·3 M ammonium sulphate 0·05 M Tris-H2SO4 pH 8·5. The crystals diffract well to at least a resolution of 1·8 A. There are 6 molecules in the unit cell of dimensions: a = b = 75·03 A , c = 49·04 A , γ = 120°. The space group is P312 1 or P322 1.