Bror Strandberg

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.

Crystal structure of human erythrocyte carbonic anhydrase C: III. Molecular structure of the enzyme and of one enzyme-inhibitor complex at 5.5 Å resolution☆

Abstract The structures of human carbonic anhydrase C and of the complex between the enzyme and the inhibitor acetoxymercurisulphanilamide have been determined by X-ray diffraction using four heavy-atom derivatives. The enzyme molecule is an ellipsoid with the approximate dimensions 40 A × 45 A × 55 A. At the active site the molecule has a large cavity, at the bottom of which the zinc atom is bound. One part of the cavity is a narrow slit where the sulphonamide inhibitor attach and bind to the zinc. The distance between the zinc atom and the only SH-group of the enzyme is about 14 A. The most likely polypeptide chain-folding and the helical content are discussed.

Presentation of a foreign peptide on the surface of tomato bushy stunt virus.

A 13-amino-acid peptide derived from the V3 loop of human immunodeficiency virus (HIV-1) glycoprotein 120 (gp120) was attached as a C-terminal gene fusion to the coat protein of tomato bushy stunt virus (TBSV). The architecture of this plant virus permitted external display of the foreign sequence 180 times on the surface of the chimaeric virus particle. The chimaera replicated to a level similar to wild-type TBSV and the foreign sequence was retained through six sequential passages in plants. The HIV epitope was detected on the surface of the virus capsid by a V3-specific monoclonal antibody and by human sera from HIV-1-positive patients, demonstrating the potential of using plant-derived chimaeric particles for diagnostic purposes. Chimaeric virus also induced a specific immune response to the foreign HIV epitope when injected into NMRI mice.

Structural comparisons of some small spherical plant viruses

The structures of tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been compared quantitatively. The organization of the shell domains of tomato bushy stunt virus and southern bean mosaic virus within the icosahedral envelope is identical. The wedge-shaped end of the subunit is closer to the fivefold or quasi-sixfold axes in all three viruses but the packing about the three- and twofold axes is quite different in satellite tobacco necrosis virus as compared to tomato bushy stunt virus or southern bean mosaic virus. The polypeptide folds of these viruses have greatest similarity in the beta-sheet region of the eight-stranded anti-parallel beta-barrel. The largest differences occur in the connecting segments. There is no clear indication of homologous amino acid sequences between southern bean mosaic virus and satellite tobacco necrosis virus. However, there is some conservation of the following functional groups. (1) Threonines and serines at the hexagonal-pentagonal wedge-shaped end of the subunit. (2) Lysines and arginines at the protein-RNA interface. (3) Hydrophobic residues in the cavity within the anti-parallel beta-barrel. (4) An aspartic acid near a site which binds Ca in tomato bushy stunt virus. (5) Ionic interactions in the contacts between fivefold-related subunits. These virus coat protein structures are not as similar to each other as the alpha and beta chains of hemoglobin but have greater likeness to one another than the NAD-binding domains of dehydrogenases or lysozymes from hen egg-white and T4 phage. The surface domains of tomato bushy stunt virus and southern bean mosaic virus are more like each other than like satellite tobacco necrosis virus. A divergent evolutionary tree is proposed on the basis of these observations.

Crystal structure studies on human erythrocyte carbonic anhydrase C. (II).

Several heavy-atom derivatives of human carbonic anhydrase form C have been prepared. Mercury atoms have been placed in at least four different sites. Two of the heavy-atom derivatives were obtained using sulphonamide inhibitors. The zinc atom, necessary for enzyme activity, has been removed and replaced by a mercury atom by means of reactions in the enzyme crystals. The positions of the heavy atoms and degrees of substitution in these positions were refined by a least squares procedure. For all derivatives, the mercury content found by X-ray investigation has been correlated with the values obtained by activation analyses. By using the native enzyme and four different isomorphous heavy-atom derivatives, X-ray analysis has led to an electron density projection of the unit cell of the protein. The same derivatives will be used to determine the three-dimensional structure of the enzyme (this work is in progress to 5.5 A resolution). Chemical and crystallographical evidence has led to approximate positions of the zinc atom and of the only SH-group in the enzyme molecule. The minimum Zn-S distance is 10 A. In sulphonamide derivatives one small electron density peak (probably a sulphonamide group) is situated close to the zinc position.

Crystallography in molecular biology

J.R. Helliwell+ SERC, Daresbury Laboratory Daresbury Warrington WA4 4AD, U.K. A survey is given of the data collection requirements for protein crystal structure analysis. The available hardware is reviewed including x-ray source and beam conditioning but specifically detector technology is discussed as the principal theme.

Purification, crystallization and preliminary X-ray data of the bacteriophage MS2

Single crystals of the bacteriophage MS2 have been produced by the vapour diffusion technique in the presence of 1.5% polyethylene glycol 6000 and 0.2 M-sodium phosphate buffer (pH 7.4). These are the first bacteriovirus crystals diffracting to high resolution. The crystal space group is C2 with the unit cell parameters a = 467.9 A, b = 289.5 A, c = 275.6 A and beta = 121.8 degrees. The asymmetric unit contains one half of the virion. The maximum resolution limit of the X-ray diffraction data obtained from these crystals was 2.9 A. The purification of the virus material was done by mild procedures exclusively and involved precipitation with polyethylene glycol 6000 and size exclusion chromatography on Sepharose CL-4B.

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.

Immunological Characterization Of The Human Immunodeficiency Virus Type 1 Reverse Transcriptase Protein By The Use Of Monoclonal Antibodies

Eighteen monoclonal antibodies (MAbs) directed against the purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) protein were produced. The antibodies were characterized by competitive ELISAs and Western blot experiments, and with nested, nine amino acid long peptides representing the whole 560 amino acid RT protein. By ELISA, the MAbs react with a minimum of seven epitopes of the protein. Four of the epitopes were located on the N-terminal 51K subunit and the remaining three epitopes were located at the C-terminal end of the protein. Using synthetic peptides, two epitopes at the N-terminal part were located at amino acids 294 to 302 and 350 to 354, respectively, from the N-terminal start of the protein. One epitope was located at amino acids 442 to 450, just after the cleavage site between the N-terminal and C-terminal subunit at position 440. Antibodies located at amino acids 294 to 302 could inhibit the RT enzymic activity of the protein. Two other MAbs, directed at the N-terminal and C-terminal parts of the protein, could also inhibit RT activity.

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.