Docking phospholipase A2 on membranes using electrostatic potential- modulated spin relaxation magnetic resonance

Science

Volumn 279, Issue 5358, 1998, Pages 1925-1929

  Lin, Ying ^a   Nielsen, Robert ^a   Murray, Diana ^b   Hubbell, Wayne L ^c   Mailer, Colin ^a   Robinson, Bruce H ^a   Gelb, Michael H ^a  

^a UNIVERSITY OF WASHINGTON   (United States)

^b STONY BROOK UNIVERSITY   (United States)

^c JULES STEIN EYE INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BEE VENOM; CHROMIUM; OXALIC ACID; PHOSPHOLIPASE A2; CHROMIC ACID; DIMYRISTOYLMETHYLPHOSPHATIDIC ACID; GLYCEROPHOSPHOLIPID; LIPOSOME; MEMBRANE PROTEIN; OXALIC ACID DERIVATIVE; PHOSPHATIDIC ACID; PHOSPHOLIPASE A; TRIS(OXALATO)CHROMATE(III);

ARTICLE; ELECTRON SPIN RESONANCE; MEMBRANE BINDING; PRIORITY JOURNAL; SPIN LABELING; ARTIFICIAL MEMBRANE; BINDING SITE; CHEMICAL STRUCTURE; CHEMISTRY; GENETICS; METABOLISM; MUTATION; SURFACE PROPERTY;

BEE VENOMS; BINDING SITES; CHROMATES; ELECTRON SPIN RESONANCE SPECTROSCOPY; GLYCEROPHOSPHOLIPIDS; LIPOSOMES; MEMBRANE PROTEINS; MEMBRANES, ARTIFICIAL; MODELS, MOLECULAR; MUTATION; OXALATES; PHOSPHATIDIC ACIDS; PHOSPHOLIPASES A; SPIN LABELS; SURFACE PROPERTIES;

EID: 0032549181     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5358.1925     Document Type: Article

References (46)

1. D. L. Scott and P. B. Sigler, Adv. Protein Chem. 45, 53 (1994); D. W. Heinz, M. Ryan, T. L. Bullock, O. H. Griffith, EMBO J. 14, 3855 (1995); L. O. Essen, O. Perisic, R. Cheung, M. Katan, R. L. Williams, Nature 380, 595 (1996); H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, ibid. 359, 159 (1992); Z. S. Derewenda, Adv. Protein Chem. 45, 1 (1994).
2. D. L. Scott and P. B. Sigler, Adv. Protein Chem. 45, 53 (1994); D. W. Heinz, M. Ryan, T. L. Bullock, O. H. Griffith, EMBO J. 14, 3855 (1995); L. O. Essen, O. Perisic, R. Cheung, M. Katan, R. L. Williams, Nature 380, 595 (1996); H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, ibid. 359, 159 (1992); Z. S. Derewenda, Adv. Protein Chem. 45, 1 (1994).
3. D. L. Scott and P. B. Sigler, Adv. Protein Chem. 45, 53 (1994); D. W. Heinz, M. Ryan, T. L. Bullock, O. H. Griffith, EMBO J. 14, 3855 (1995); L. O. Essen, O. Perisic, R. Cheung, M. Katan, R. L. Williams, Nature 380, 595 (1996); H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, ibid. 359, 159 (1992); Z. S. Derewenda, Adv. Protein Chem. 45, 1 (1994).
4. D. L. Scott and P. B. Sigler, Adv. Protein Chem. 45, 53 (1994); D. W. Heinz, M. Ryan, T. L. Bullock, O. H. Griffith, EMBO J. 14, 3855 (1995); L. O. Essen, O. Perisic, R. Cheung, M. Katan, R. L. Williams, Nature 380, 595 (1996); H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, ibid. 359, 159 (1992); Z. S. Derewenda, Adv. Protein Chem. 45, 1 (1994).
5. D. L. Scott and P. B. Sigler, Adv. Protein Chem. 45, 53 (1994); D. W. Heinz, M. Ryan, T. L. Bullock, O. H. Griffith, EMBO J. 14, 3855 (1995); L. O. Essen, O. Perisic, R. Cheung, M. Katan, R. L. Williams, Nature 380, 595 (1996); H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, ibid. 359, 159 (1992); Z. S. Derewenda, Adv. Protein Chem. 45, 1 (1994).
6. M. M. G. M. Thunnissen et al., Nature 347, 689 (1990); D. L. Scott et al., Science 250, 1541 (1990); B. W. Dijkstra, R. Renetseder, K. H. Kalk, W. G. Hol, J. Drenth, J. Mol. Biol. 168, 163 (1983); A. Achari et al., Cold Spring Habor Symp. Quant. Biol. 52, 441 (1987).
7. M. M. G. M. Thunnissen et al., Nature 347, 689 (1990); D. L. Scott et al., Science 250, 1541 (1990); B. W. Dijkstra, R. Renetseder, K. H. Kalk, W. G. Hol, J. Drenth, J. Mol. Biol. 168, 163 (1983); A. Achari et al., Cold Spring Habor Symp. Quant. Biol. 52, 441 (1987).
8. M. M. G. M. Thunnissen et al., Nature 347, 689 (1990); D. L. Scott et al., Science 250, 1541 (1990); B. W. Dijkstra, R. Renetseder, K. H. Kalk, W. G. Hol, J. Drenth, J. Mol. Biol. 168, 163 (1983); A. Achari et al., Cold Spring Habor Symp. Quant. Biol. 52, 441 (1987).
9. M. M. G. M. Thunnissen et al., Nature 347, 689 (1990); D. L. Scott et al., Science 250, 1541 (1990); B. W. Dijkstra, R. Renetseder, K. H. Kalk, W. G. Hol, J. Drenth, J. Mol. Biol. 168, 163 (1983); A. Achari et al., Cold Spring Habor Symp. Quant. Biol. 52, 441 (1987).
10. C. Altenbach, T. Marti, H. G. Khorana, W. L. Hubbell, Science 248, 1088 (1990); C. Altenbach, D. A. Greenhalgh, H. G. Khorana, W. L. Hubbell, Proc. Natl. Acad. Sci. U.S.A. 91, 1667 (1994).
11. C. Altenbach, T. Marti, H. G. Khorana, W. L. Hubbell, Science 248, 1088 (1990); C. Altenbach, D. A. Greenhalgh, H. G. Khorana, W. L. Hubbell, Proc. Natl. Acad. Sci. U.S.A. 91, 1667 (1994).
12. 1% = 13 cm-1) in a Speed-Vac (Savant) and stored at 4°C for several months without loss of enzymatic activity. The specific enzymatic activities of all spin-labeled mutants except I78C-sl are within a factor of 2 of that of wild-type enzyme when assayed fluorimetrically with anionic vesicles [T. Bayburt et al., Anal Biochem. 232, 7 (1995)]. The specific activity of I78C-sl was only 7% of the wild-type value.
13. 1% = 13 cm-1) in a Speed-Vac (Savant) and stored at 4°C for several months without loss of enzymatic activity. The specific enzymatic activities of all spin-labeled mutants except I78C-sl are within a factor of 2 of that of wild-type enzyme when assayed fluorimetrically with anionic vesicles [T. Bayburt et al., Anal Biochem. 232, 7 (1995)]. The specific activity of I78C-sl was only 7% of the wild-type value.
14. 1% = 13 cm-1) in a Speed-Vac (Savant) and stored at 4°C for several months without loss of enzymatic activity. The specific enzymatic activities of all spin-labeled mutants except I78C-sl are within a factor of 2 of that of wild-type enzyme when assayed fluorimetrically with anionic vesicles [T. Bayburt et al., Anal Biochem. 232, 7 (1995)]. The specific activity of I78C-sl was only 7% of the wild-type value.
15. 2 and 22 to 27 mM DTPM as small unilamellar vesicles prepared by sonication [M. K. Jain and M. H. Gelb, Methods Enzymol. 197, 112 (1991)]. EPR spectra were acquired with a resolution of 0.125 G per point and were averaged three times [C. Mailer, S. J. Danielson, B. H. Robinson, Rev. Sci. Instrum. 56, 1917 (1995); C. Mailer, D. A. Haas, E. J. Hustedt, J. G. Gladden, B. H. Robinson, J. Magn. Reson. 91, 475 (1991)].
16. 2 and 22 to 27 mM DTPM as small unilamellar vesicles prepared by sonication [M. K. Jain and M. H. Gelb, Methods Enzymol. 197, 112 (1991)]. EPR spectra were acquired with a resolution of 0.125 G per point and were averaged three times [C. Mailer, S. J. Danielson, B. H. Robinson, Rev. Sci. Instrum. 56, 1917 (1995); C. Mailer, D. A. Haas, E. J. Hustedt, J. G. Gladden, B. H. Robinson, J. Magn. Reson. 91, 475 (1991)].
17. 2 and 22 to 27 mM DTPM as small unilamellar vesicles prepared by sonication [M. K. Jain and M. H. Gelb, Methods Enzymol. 197, 112 (1991)]. EPR spectra were acquired with a resolution of 0.125 G per point and were averaged three times [C. Mailer, S. J. Danielson, B. H. Robinson, Rev. Sci. Instrum. 56, 1917 (1995); C. Mailer, D. A. Haas, E. J. Hustedt, J. G. Gladden, B. H. Robinson, J. Magn. Reson. 91, 475 (1991)].
18. F. Ghomashchi, B. Z. Yu, M. K. Jain, M. H. Gelb, Biochemistry 30, 9559 (1991).
19. C. Altenbach, S. L. Flitsch, H. G. Khorana, W. L. Hubbell, ibid. 28, 7806 (1989); D. A. Haas, C. Mailer, B. H. Robinson, Biophys. J. 64, 594 (1993).
20. C. Altenbach, S. L. Flitsch, H. G. Khorana, W. L. Hubbell, ibid. 28, 7806 (1989); D. A. Haas, C. Mailer, B. H. Robinson, Biophys. J. 64, 594 (1993).
21. C. Altenbach, W. Froncisz, J. S. Hyde, W. L. Hubbell, Biophys. J. 56, 1183 (1989).
22. Y. N. Molin, K. M. Salikhov, K. I. Zamaraev, Spin Exchange: Principles and Applications in Chemistry and Biology (Springer-Verlag, New York, 1980); J. S. Hyde, H. M. Shartz, W. E. Antholine, in Spin Labeling II: Theory and Applications L. J. Berliner, Eds. (Academic Press, New York, 1979), pp. 71-113.
23. Y. N. Molin, K. M. Salikhov, K. I. Zamaraev, Spin Exchange: Principles and Applications in Chemistry and Biology (Springer-Verlag, New York, 1980); J. S. Hyde, H. M. Shartz, W. E. Antholine, in Spin Labeling II: Theory and Applications L. J. Berliner, Eds. (Academic Press, New York, 1979), pp. 71-113.
24. 2 are typically ±5% or less and a few are ±10% or less. Numbers in parentheses were obtained with 10 mM Ni(ethylenediaminoediacetic acid) instead of Crox. I2C-sl 0.81, 0.134, 0.75, 0.136, 1.05, 0.141, 0.53, 0.42; N13C-sl 0.73 (0.85), 0.056 (0.097), 0.77 (0.78), 0.18 (0.32), 1.045 (1.045), 0.098 (0.098), 0.79 (0.78), 0.42 (0.384); K14C-sl 0.97, 0.124, 0.92, 0.130, 1.06, 0.1101, 0.78, 0.316; S15C-sl 1.02 (1.02), 0.127 (0.127), 0.82 (0.67), 0.159 (0.227), 1.06 (1.06), 0.115 (0.115), 0.81 (0.86), 0.307 (0.42); R23C-sl 0.74, 0.082, 0.78, 0.228, 1.08, 0.081, 0.83, 0.54; F24C-sl 0.72, 0.077, 0.69, 0.19, 0.99, 0.082, 0.52, 0.51; T51C-sl 0.94, 0.094, 0.99, 0.24, 1.32, 0.206, 1.06, 0.70; T53C-sl 0.92, 0.043, 0.86, 0.200, 0.92, 0.079, 0.97, 0.611; K66C-sl 0.92, 0.110, 0.92, 0.39, 1.17, 0.117, 0.86, 0.45; I78C-sl 0.81, 0.157, 0.70, 0.162, 0.96, 0.092, 0.70, 0.72; F82C-sl 0.93, 0.091, 0.87, 0.14, 0.96, 0.1003, 0.65, 0.28; K85C-sl 0.86, 0.07, 1.01, 0.143, 0.67, 0.032, 0.64, 0.15; D92C-sl 0.93, 0.038, 1.05, 0.164, 1.05, 0.164, 1.04, 0.0875, 0.86, 0.38.
25. 2] is rather uniform, even through the outer surfaces of the membrane [C. Altenbach, D. A. Greenhaigh, H. G. Khorana, W. L. Hubbell, Proc. Nat. Acad. Sci. U.S.A. 91, 1667 (1994)], and thus the major changes are expected to occur because of motional effects [B. H. Robinson, D. A. Haas, C. Mailer, Science 261, 490 (1994)]. These arguments suggest that structural rearrangements due to the membrane binding cause minimal changes in the reporter groups; the major change comes from altered local dynamics of the spin probe. More important, it can be assumed that χ (Eqs. 3 and 5) is independent of membrane binding, which depends primarily on the translational diffusion coefficient of Crox.
26. 2] is rather uniform, even through the outer surfaces of the membrane [C. Altenbach, D. A. Greenhaigh, H. G. Khorana, W. L. Hubbell, Proc. Nat. Acad. Sci. U.S.A. 91, 1667 (1994)], and thus the major changes are expected to occur because of motional effects [B. H. Robinson, D. A. Haas, C. Mailer, Science 261, 490 (1994)]. These arguments suggest that structural rearrangements due to the membrane binding cause minimal changes in the reporter groups; the major change comes from altered local dynamics of the spin probe. More important, it can be assumed that χ (Eqs. 3 and 5) is independent of membrane binding, which depends primarily on the translational diffusion coefficient of Crox.
27. N. Lakshminarayanaiah, Equations of Membrane Biophysics (Academic Press, Orlando, FL, 1984).
28. 15N species was calculated. The ratio of exchange rates for the two relaxing agents of differing charge is equal to the ratio of local concentrations of these agents at the lipidated spin label location, and thus ψ(0) may be determined [Y.-K. Shin and W. Hubbell, Biophys. J. 61, 1443 (1992); J. Castle and W. Hubbell, Biochemistry 15, 4818 (1976)].
29. 15N species was calculated. The ratio of exchange rates for the two relaxing agents of differing charge is equal to the ratio of local concentrations of these agents at the lipidated spin label location, and thus ψ(0) may be determined [Y.-K. Shin and W. Hubbell, Biophys. J. 61, 1443 (1992); J. Castle and W. Hubbell, Biochemistry 15, 4818 (1976)].
30. 15N species was calculated. The ratio of exchange rates for the two relaxing agents of differing charge is equal to the ratio of local concentrations of these agents at the lipidated spin label location, and thus ψ(0) may be determined [Y.-K. Shin and W. Hubbell, Biophys. J. 61, 1443 (1992); J. Castle and W. Hubbell, Biochemistry 15, 4818 (1976)].
31. 15N species was calculated. The ratio of exchange rates for the two relaxing agents of differing charge is equal to the ratio of local concentrations of these agents at the lipidated spin label location, and thus ψ(0) may be determined [Y.-K. Shin and W. Hubbell, Biophys. J. 61, 1443 (1992); J. Castle and W. Hubbell, Biochemistry 15, 4818 (1976)].
32. D. L. Scott, Z. Otwinowski, M. H. Gelb, P. B. Sigler, Science 250, 1563 (1990).
33. W. H. Press, B. P. Flannery, S. A. Teukolsky, W. T. Vetterling, Numerical Recipes (Cambridge Univ. Press, Cambridge, 1987).
34. 2 by avoiding van der Waals conflict and by using reasonable dihedral angles including -90 for the S-S dihedral angle. Because there is some uncertainty in the placement of the spin labels, the regression fit of the modeled structure to the EPR results was repeated after one spin label at a time was omitted from the set. The changes in orientation and in distance from the membrane in all cases were within the errors given by the fit with all spin labels included. The spin labels at positions 14, 15, and 23 were removed as a set because they were deemed to have the most uncertainty in spin-labeling position. Again, the fit was within the stated errors. The good observed fit is due in part to the fact that 13 spin labels were used, and there are not just one or two residues that dictate the structure.
35. Crox, ψ(0) and a convolution over ψ(r), only affected the protein-membrane distance and did not alter the protein-membrane angular orientation. A third Euler angle is not needed because the membrane is of infinite extent. The Euler angles and protein-membrane distance have errors of less than 10° and 2 Å, respectively, as given by the standard error of propagation for nonlinear least squares algorithms. The data for I78C-sl were not included in the fit because they are clearly anomalous (Table 1). Because this mutant has only 7% of the enzymatic activity of the wild type, EPR data are not useful. All fitting was done with MATLAB (Mathworks, Cambridge, MA).
36. Because the Crox-nitroxide spin exchange happens as fast as Crox diffuses to the nitroxide (9), the possibility that the presence of the membrane slows the rate of spin relaxation by slowing the rate of diffusion-limited Crox-nitroxide encounters was also considered, but the calculation (available from the authors on request) shows that diffusional effects only occur if the membrane is <3 Å from the spin label, and thus diffusional effects are not responsible for the deviation of Φ from unity measured when the spin label is tens of angstroms away from the membrane.
37. M). The calculated potential differences (residue and average ± SD in millivolts) are: 2, -26 ± 15; 13, -7 ± 5; 14, -16 ± 10; 15, -6 ± 8; 23, -12 ± 6; 24, -9 ± 3; 51, -2 ± 3; 53, 0 ± 1; 66, 3 ± 1; 78, -9 ± 3; 85, -6 ± 2; and 92, -3 ± 1.
38. B. Honig and A. Nicholls, Science 268, 1144 (1995).
39. T. Thuren et al., Biochemistry 23, 5129 (1984).
40. M. K. Jain, B.-Z. Yu, J. Rogers, G. N. Ranadive, O. Berg, Biochemistry 30, 7306 (1991); B.-Z. Yu et al., ibid. 36, 3870 (1997).
41. M. K. Jain, B.-Z. Yu, J. Rogers, G. N. Ranadive, O. Berg, Biochemistry 30, 7306 (1991); B.-Z. Yu et al., ibid. 36, 3870 (1997).
42. F. Ghomashchi et al., ibid., in press.
43. N. Din et al., Mol. Microbiol. 11, 747 (1994).
44. P. Kraulis, J. Appl. Crystallogr. 24, 946 (1991); E. A. Merritt and D. J. Bacon, Methods Enzymol. 277, 505 (1997).
45. P. Kraulis, J. Appl. Crystallogr. 24, 946 (1991); E. A. Merritt and D. J. Bacon, Methods Enzymol. 277, 505 (1997).
46. 15N spin labels.