- Email: [email protected]
Effect of β-D-octyl glucoside on protein crystal habit
150
Second American Symposium
The crystals grow as rectangular plates, occasionally as large as 0.8 x 0.5 x 0.2 mm . They diffract to a resolution better than 2.5 A, exhibit space group symmetry P2,2,2, and have lattice rnnstants a = 38 .6, b = 69 .9 and c = 77 .6 A. There is a single heterodimer in theasymalaric unit . These crystals are thus excellent candidates for a successful, single crystal x-ray diffraction analysis . Data collection is in progress and we intend to use the isomorphous replacement technique for analysis, as well as molecular replacemrnt methods based on the presumed structural homology between the basic subunit and crotalid phospholipase A, . Currrnt results of these analyses will be presented. A1RD, S. D. and KAISER, I. I . (1985)
Toxicon 23,
361.
F,(jeet ojß-D-octyJ glutaride on protein crystal habit . TRINA M. NORDEN and KEITH B. WARD (Laboratory for the Structure of Matter Naval Research Laboratory, Washington, DC, U.S .A .). To BE surrAatE for x-ray diffraction analysis, single crystals of protein should exhibit an equidimrnsional or 'chunky' habit, so that approximately equal crystal volumes will be immersed in the incidrnt beam at all crystal orientations. To improve the crystal habit of cardiotoxin III from the venom of Ngja ngja arcs (mol . wt = 6700) we Gave begun experimrnts which have tested the influence of ß-D-0ctyl glucoside on crystal formation. This amphipathic molecule is commonly used in the crystalliTation of membrane proteins (GARAVITO and ROBENBUSCH, 1980 ; Mlct~t and OESTERxetT, 1980) and is reported to effect the crystal formation of globular proteins (McPItt:RSON et al., 1986). Our initial results are encouraging and are described below . Cardiotoxin III protein solution wan combined with ß-D-octyl glucoside and stored at 4°C for 4 hr. The hanging droplets were thrn created by mixing betwern 1 and 4 ~1 of the reservoir solution and 10 ~l of the protein/ß-D-octyl glucoside solution at room temperature. The reservoir solution in every case was 1 .0 M phosphate buffer, pH 7.0 . The droplets were of initial protein concentration betwern 8.18 and 5 .00 mg/ml and had an initial ß-D-octyl glucoside concentration ranging from 0.07 to 0.424'0 . Cardiotoxin III crystals grown exclusively in the presence of phosphate buffer yielded long, thin needles. At the lowest conantrationa of ß-D-octyl glucoside these needles are much shorter and thinner. However, as the initial concentration reached approximately 0.24'0 ß-D-octyl glucoside, thin plates became interspersed with the :mall, thin needles. As the initial concentration of ß-D-0ctyl glucoside increased further, plates were the only crystal form present in the droplets . An inaease in the length of time allowed for the combination of the protein with ß-D-octyl glucoside before setting up the hanging drops did not appear to greatly affect the resulting crystals. Further studies involving the use of ß-D-octyl glucoside at higher concentrations and with other proteins are currently under investigation . In this way we hope to prepare single crystals of several protein toxins suitable for x-ray diffraction analysis . CiAawvrro, M. and ROSENBUSCFI, J. P. (1980) J. Cel! Biol. 86, 327. MCPHERSON, A., KOSZELAK, S., AXELROD, H., DAY, l., WILLIAMS, R., ROBINSON, L., MCGRATH, M . and CASCIO, D. (1986) J. biol. Chem . 261, 1969. Mlctmt, H. and OESCERHELT, D. (1980) Proc. natn. Acad. Sci. U.S.A . 77, 1283 .
The ribosome-inactivating rntalytic protein toxins: a comparison of their biological properties. ToM G. OBRIc (Departmrnt of Microbiology and Immunology, Albany Medical College, Albany, New York 12208, U.S.A.) . A CLASS of toxic proteins exists with members having in common the ability to selectively inhibit protein biosynthesis in eukaryotes . These proteins are unique in that they all inactivate cytoplasmic ribosomes of eukaryotic cells in a catalytic manner . Further, they arc without effect on prokaryotic ribosomes and protein synthesis. Proteins of this group are produced by higher plants (ricin, phytolaccin, abrin), fungi (alpha-sarcin) and bacteria (Shiga toxin). Structurally, these individual holotoxins differ considerably, ranging from a single A subunit (e .g. phytolaain) to a complex of one A subunit and 6 B subunts (Shiga toxin) . The A and B aubunits represent the catalytic and cell sodas-binding moieties, respectively. Interestingly, the A subunits of these different toxins are antigrnically distinct, despite their having a similar mode of action . Differences in the ribosome-inactivating process are suggested by resent data for Shiga toxin, phytolaccin and alpha-sarcin . While these three proteins possess ribonuclease activity with naked rRNA substrates, only alpha-sarcin specifically cleaves a Ltrge RNA fragmrnt from the 28S rRNA of intact 606 ribosomes . Moreover, the functional lesion in protein synthesis dlffera following treatment of rrbosomma with either Shiga toxin, phytolaccin or alpha-aarcin . Ribosomes treated with Shiga toxin appear to be inhibited at the elongation factor 1-deprndrnt step of protein synthesi:, while phytoLuxdtl appears to preferentially inhibit elongation factor Jrdeprndrnt reactions. Alphaeardn affects EF-1 reactions and is unique in its ability to slow peptide initiation . Differrntial properties of the A
Second American Symposium
The crystals grow as rectangular plates, occasionally as large as 0.8 x 0.5 x 0.2 mm . They diffract to a resolution better than 2.5 A, exhibit space group symmetry P2,2,2, and have lattice rnnstants a = 38 .6, b = 69 .9 and c = 77 .6 A. There is a single heterodimer in theasymalaric unit . These crystals are thus excellent candidates for a successful, single crystal x-ray diffraction analysis . Data collection is in progress and we intend to use the isomorphous replacement technique for analysis, as well as molecular replacemrnt methods based on the presumed structural homology between the basic subunit and crotalid phospholipase A, . Currrnt results of these analyses will be presented. A1RD, S. D. and KAISER, I. I . (1985)
Toxicon 23,
361.
F,(jeet ojß-D-octyJ glutaride on protein crystal habit . TRINA M. NORDEN and KEITH B. WARD (Laboratory for the Structure of Matter Naval Research Laboratory, Washington, DC, U.S .A .). To BE surrAatE for x-ray diffraction analysis, single crystals of protein should exhibit an equidimrnsional or 'chunky' habit, so that approximately equal crystal volumes will be immersed in the incidrnt beam at all crystal orientations. To improve the crystal habit of cardiotoxin III from the venom of Ngja ngja arcs (mol . wt = 6700) we Gave begun experimrnts which have tested the influence of ß-D-0ctyl glucoside on crystal formation. This amphipathic molecule is commonly used in the crystalliTation of membrane proteins (GARAVITO and ROBENBUSCH, 1980 ; Mlct~t and OESTERxetT, 1980) and is reported to effect the crystal formation of globular proteins (McPItt:RSON et al., 1986). Our initial results are encouraging and are described below . Cardiotoxin III protein solution wan combined with ß-D-octyl glucoside and stored at 4°C for 4 hr. The hanging droplets were thrn created by mixing betwern 1 and 4 ~1 of the reservoir solution and 10 ~l of the protein/ß-D-octyl glucoside solution at room temperature. The reservoir solution in every case was 1 .0 M phosphate buffer, pH 7.0 . The droplets were of initial protein concentration betwern 8.18 and 5 .00 mg/ml and had an initial ß-D-octyl glucoside concentration ranging from 0.07 to 0.424'0 . Cardiotoxin III crystals grown exclusively in the presence of phosphate buffer yielded long, thin needles. At the lowest conantrationa of ß-D-octyl glucoside these needles are much shorter and thinner. However, as the initial concentration reached approximately 0.24'0 ß-D-octyl glucoside, thin plates became interspersed with the :mall, thin needles. As the initial concentration of ß-D-0ctyl glucoside increased further, plates were the only crystal form present in the droplets . An inaease in the length of time allowed for the combination of the protein with ß-D-octyl glucoside before setting up the hanging drops did not appear to greatly affect the resulting crystals. Further studies involving the use of ß-D-octyl glucoside at higher concentrations and with other proteins are currently under investigation . In this way we hope to prepare single crystals of several protein toxins suitable for x-ray diffraction analysis . CiAawvrro, M. and ROSENBUSCFI, J. P. (1980) J. Cel! Biol. 86, 327. MCPHERSON, A., KOSZELAK, S., AXELROD, H., DAY, l., WILLIAMS, R., ROBINSON, L., MCGRATH, M . and CASCIO, D. (1986) J. biol. Chem . 261, 1969. Mlctmt, H. and OESCERHELT, D. (1980) Proc. natn. Acad. Sci. U.S.A . 77, 1283 .
The ribosome-inactivating rntalytic protein toxins: a comparison of their biological properties. ToM G. OBRIc (Departmrnt of Microbiology and Immunology, Albany Medical College, Albany, New York 12208, U.S.A.) . A CLASS of toxic proteins exists with members having in common the ability to selectively inhibit protein biosynthesis in eukaryotes . These proteins are unique in that they all inactivate cytoplasmic ribosomes of eukaryotic cells in a catalytic manner . Further, they arc without effect on prokaryotic ribosomes and protein synthesis. Proteins of this group are produced by higher plants (ricin, phytolaccin, abrin), fungi (alpha-sarcin) and bacteria (Shiga toxin). Structurally, these individual holotoxins differ considerably, ranging from a single A subunit (e .g. phytolaain) to a complex of one A subunit and 6 B subunts (Shiga toxin) . The A and B aubunits represent the catalytic and cell sodas-binding moieties, respectively. Interestingly, the A subunits of these different toxins are antigrnically distinct, despite their having a similar mode of action . Differences in the ribosome-inactivating process are suggested by resent data for Shiga toxin, phytolaccin and alpha-sarcin . While these three proteins possess ribonuclease activity with naked rRNA substrates, only alpha-sarcin specifically cleaves a Ltrge RNA fragmrnt from the 28S rRNA of intact 606 ribosomes . Moreover, the functional lesion in protein synthesis dlffera following treatment of rrbosomma with either Shiga toxin, phytolaccin or alpha-aarcin . Ribosomes treated with Shiga toxin appear to be inhibited at the elongation factor 1-deprndrnt step of protein synthesi:, while phytoLuxdtl appears to preferentially inhibit elongation factor Jrdeprndrnt reactions. Alphaeardn affects EF-1 reactions and is unique in its ability to slow peptide initiation . Differrntial properties of the A