Precise boundary element computation of protein transport properties: Diffusion tensors, specific volume, and hydration

Biophysical Journal

Volumn 91, Issue 5, 2006, Pages 1591-1603

  Aragon, Sergio ^a   Hahn, David K ^a  

^a SAN FRANCISCO STATE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CRYSTALLOGRAPHY; DIFFUSION COEFFICIENT; DIFFUSION TENSOR IMAGING; HYDRODYNAMICS; MACROMOLECULE; MATHEMATICAL COMPUTING; PROTEIN ANALYSIS; PROTEIN FUNCTION; PROTEIN STRUCTURE; PROTEIN TRANSPORT;

EID: 33748451514     PISSN: 00063495     EISSN: None     Source Type: Journal    
DOI: 10.1529/biophysj.105.078188     Document Type: Article

References (131)

1. Giacovazzo, C. 1992. Fundamentals of Crystallography. Oxford University Press, New York.
2. Richards, E. G. 1980. An Introduction to Physical Properties of Large Molecules in Solution. Cambridge University Press, New York.
3. Berne, B., and R. Pecora. 1976. Dynamic Light Scattering: With applications to Chemistry, Biology and Physics. Wiley-Interscience, New York.
4. Eden, D., and J. G. Elias. 1983. Transient electric birefringence of DNA restriction fragments and the filamentous virus Pf3. In Measurement of Suspended Particles by Quasi-Elastic Light Scattering. B. Dahneke, editor. Wiley-Interscience, New York. 401-438.
5. Stryer, L. 1968. Fluorescence spectroscopy of proteins. Science. 162:526-533.
6. Swaminathan, R., C. P. Hoang, and A. S. Verkman. 1997. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys. J. 72:1900-1907.
7. Ryba, N. J. P., and D. Marsh. 1992. Protein rotational diffusion and lipid/protein interactions in recombinants of bovine rhodopsin with saturated diacylphosphatidylcholines of different chain lengths studies by conventional and saturation-transfer electron spin resonance. Biochemistry. 31:7511-7518.
8. Sanders, J. K. M., and B. K. Hunter. 1987. Modern NMR Spectroscopy. Oxford University Press, New York.
9. Harding, S. E., A. J. Rowe, and W. V. Shaw. 1987. The molecular mass and trimeric nature of chloramphenicol transacetylase. Biochem. Soc. Trans. 15:513-519.
10. Aragon, S. R. 2004. A precise boundary element method for macromolecular transport properties. J. Comput. Chem. 25:1191-1205.
11. Bloomfield, V. A., W. O. Dalton, and K. E. van Holde. 1967. Frictional coefficients of multisubunit structures. I. Theory. Biopolymers. 5:135-148.
12. Frictional coefficients of multisubunit structures. II. Application to proteins and viruses. Biopolymers. 5:149-159.
13. Garcia de la Torre, J., and V.A Bloomfield. 1981. Hydrodynamic properties of complex, rigid biological macromolecules. Theory and Applications. Quart. Rev. Biophys. 14:81-139.
14. Teller, D. C., E. Swanson, and C. de Haen. 1979. The translational friction coefficients of proteins. Methods Enzymol. 61:103-124.
15. Pastor, R. W., and M. Karplus. 1988. Parametrization of the friction constant for stochastic simulations of polymers. J. Phys. Chem. 92:2336-2341.
16. Venable, R. M., and R. W. Pastor. 1988. Frictional models for stochastic simulations of proteins. Biopolymers. 27:1001-1014.
17. Antosiewicz, J., and D. Porschke. 1989. Volume correction for bead model simulations of rotational friction coefficients of macromolecules. J. Phys. Chem. 93:5301-5305.
18. Garcia de la Torre, J., M. L. Huertas, and B. Carrasco. 2000. Calculation of hydrodynamic properties of globular proteins from their atomic-level structure. Biophys. J. 78:719-730.
19. Swanson, E., D. C. Teller, and C. de Haen. 1978. The low Reynolds number translational friction of ellipsoids, cylinders, dumbbells, and hollow spherical caps. Numerical testing of the validity of the modified Oseen tensor in computing the friction of objects modeled as beads on a shell. J. Chem. Phys. 68:5097-5102.
20. Allison, S. A. 1999. Low Reynolds number transport properties of axisymmetric particles employing stick and slip boundary conditions. Macromolecules. 32:5304-5312.
21. Allison, S. A., and V. T. Tran. 1995. Modeling the electrophoresis of rigid polyions - application to lysozyme. Biophys. J. 68:2261-2270.
22. S. A. Allison. 2001. Boundary element modeling of biomolecular transport Biophys. Chem. 93:197-213.
23. Zhou, H.-X. 1995. Calculation of translational friction and intrinsic viscosity. II. Application to globular proteins. Biophys. J. 69:2298-2303.
24. Youngren, G. K., and A. Acrivos. 1975. Stokes flow past a particle of arbitrary shape: a numerical method of solution. J. Fluid Mech. 69:377-402.
25. Pakdel, P., and S. Kim. 1991. Mobility and stresslet functions of particles with rough surfaces in viscous fluids: a numerical study. J. Reohl. 35:797-823.
26. Brune, D., and S. Kim. 1993. Predicting protein diffusion coefficients. Proc. Natl. Acad. Sci. USA. 90:3835-3839.
27. Kim, S., and S. J. Karilla. 1927. Microhydrodynamics, Butterworth-Heinemann: New York.
28. Oseen, C. W. 1927. Hydrodynamik. Academiches Verlag, Leipzig.
29. Wegener, W. A. 1986. On an exact starting expression for macromolecular hydrodynamic models. Biopolymers. 25:627-637.
30. Brenner, H. 1967. Coupling between the translational and rotational Brownian motions of rigid particles of arbitrary shape. II. General theory. Colloid Interface Sci. 23:407-436.
31. Kuntz, I. D., Jr., and W. Kauzmann. 1974. Hydration of proteins and polypeptides. Adv. Protein Chem. 28:239-345.
32. Squire, P. G., and M. E. Himmel. 1979. Hydrodynamics and protein hydration. Arch. Biochem. Biophys. 196:165-177.
33. Rupley, J. A., and G. Careri. 1991. Protein hydration and function. Adv. Protein Chem. 41:37-172.
34. Connolly, M. L. 1993. The molecular surface package. J. Mol. Graph. 11:139-141.
35. Connolly, M. L. 1983. Analytical molecular surface calculation. J. Appl. Crystallogr. 16:548-558.
36. Connolly, M. L. 1983. Solvent-accessible surfaces of proteins and nucleic acids. Science. 221:709-713.
37. Bull, K., and H. B. Breese. 1968. Protein hydration. II. Specific heat of egg albumin. Arch. Biochem. Biophys. 128:497-502.
38. Dubin, S. B., N. A. Clark, and G. B. Benedek. 1971. Measurement of the rotational diffusion coefficient of lysozyme by depolarized light scattering: configuration of lysozyme in solution. J. Chem. Phys. 54:5158-5164.
39. Sophianopoulos, A. J., C. K. Rhodes, D. N. Holcomb, and K. E. van Holde. 1962. Physical studies of lysozyme. I. Characterization. J. Biol. Chem. 237:1107-1112.
40. Irwin, R., and J. E. Churchich. 1971. Rotational relaxation time of pyridoxyl 5-phosphate lysozyme. J. Biol. Chem. 246:5329-5334.
41. Zhou, H.-X. 2001. A unified picture of protein hydration. Biophys. Chem. 93:171-179.
42. James, T. L., G. B. Matson, and I. D. Kuntz. 1978. Protein rotational correlation times determined in aqueous solution by carbon-13 rotating frame spin-lattice relaxation in the presence of an off-resonance radiofrequency field. J. Am. Chem. Soc. 100:3590-3594.
43. Riveros-Moreno, V., and J. B. Wittenberg. 1972. The self-diffusion coefficients of myoglobin and hemoglobin in concentrated solutions. J. Biol. Chem. 247:895-901.
44. South, G. P., and G. H. Grant. 1972. Dielectric dispersion and dipole moment of myoglobin in water. Proc. R. Soc. London A. 328:371-387.
45. Creeth, J. M. 1958. Studies of free diffusion in liquids with the Rayleigh method. III. The analysis of known mixtures and some preliminary investigations with proteins. J. Phys. Chem. 62:66-74.
46. Krause, S., and C. T. O'Konski. 1963. Electric properties of macromolecules. VIII. Kerr constants and rotational diffusion of some proteins in water and in glycerol-water solutions. Biopolymers. 1:503-515.
47. Richards, E. G. 1980. An introduction to physical properties of large molecules in solution. Cambridge University Press, London.
48. Parak, F. 1986. Correlation of protein dynamics with water mobility: Mossbauer spectroscopy and microwave absorption methods. Methods Enzymol. 127:196-206.
49. Durchschlag, H., and P. Zipper. 1997. Calculation of hydrodynamic parameters of biopolymers from scattering data using whole-body approaches. Prog. Colloid Polym. Sci. 107:43-57.
50. Tietze, F. 1953. Molecular-kinetic properties of crystalline trypsinogen. J. Biol. Chem. 204:1-11.
51. Takagi, T., and T. Isemura. 1966. Extent of renaturation of reduced Taka-Amylase A before reformation of disulfide bonds. Biochim. Biophys. Acta. 130:233-240.
52. Svedburg, T., and K. O. Pedersen. 1940. The Ultracentrifuge. Oxford University Press, London.
53. Altman, P. L., and D. S. Dittmer. 1972. Biology Data Book I, 2nd ed. FASEB, Bethesda, MD.
54. Wu, J.-Y., and J. T. Yang. 1970. Physicochemical characterization of citrate synthase and its subunits. J. Biol. Chem. 245:212-218.
55. Pessen, H., and T. F. Kumonski. 1985. Measurements of protein hydration by various techniques. Methods Enzymol. 117:219-257.
56. Durchschlag, H., P. Zipper, G. Purr, and R. Jaenicke. 1996. Comparative studies of structural properties and conformational changes of proteins by analytical ultracentrifugation and other techniques. Colloid Polym. Sci. 274:117-137.
57. Schwan, H. P. 1965. Electrical properties of bound water. Ann. N. Y. Acad. Sci. 125:344-354.
58. Gallagher, W. H., and C. K. Woodward. 1989. The concentration dependence of the diffusion coefficient for bovine pancreatic trypsin inhibitor: a dynamic light scattering study of a small protein. Biopolymers. 28:2001-2024.
59. Noelken, M. E., P. J. Chang, and J. R. Kimmel. 1980. Reversible dimerization of avian pancreatic polypeptide. Biochemistry. 19:1838-1843.
60. Fling, M., N. H. Horowitz, and S. F. Heinemann. 1963. The isolation and properties of crystalline tyrosinase from neurospora. J. Biol. Chem. 238:2045-2053.
61. Larew, L., and R. W. Walters. 1987. A kinetic, chromatographic method for studying protein hydrodynamic behavior. Anal. Biochem. 164:537-546.
62. Walters, R. W., J. F. Graham, R. M. Moore, and D. J. Anderson. 1984. Protein diffusion coefficient measurements by laminar flow analysis: method and applications. Anal. Biochem. 140:190-195.
63. Clark, S. M., D. G. Leaist, and L. Konermann. 2002. Taylor dispersion monitored by electrospray mass spectrometry: a novel approach for studying diffusion in solution. Rapid Commun. Mass. Spectrom. 16:1454-1462.
64. Polson, A. 1939. Über die berechnung der gestalt von proteinmolekülen. Kolloid. Z. 88:51-61.
65. Gordon, W. G., and W. F. Semmett. 1953. Isolation of crystalline α-lactalbumin from milk. J. Am. Chem. Soc. 75:328-330.
66. Patkowski, A., J. Seils, F. Buß, B. M. Jockusch, and T. Dorfmüller. 1990. Size and shape parameters of the actin-binding protein profilin in solution. A depolarized and polarized dynamic light scattering study. Biopolymers. 30:219-222.
67. Ehrenberg, A. 1957. Determination of molecular weights and diffusion coefficients in the ultracentrifuge. Acta Chem. Scand. 11:1257-1270.
68. Broughton, W. J., M. J. Dilworth, and C. A. Godfrey. 1972. Molecular properties of lupin and serradella leghaemoglobins. Biochem. J. 127:309-314.
69. Le Bon, C., T. Nicolai, M. E. Kuil, and J. G. Hollander. 1999. Self-diffusion and cooperative diffusion of globular proteins in solution. J. Phys. Chem. B. 103:10294-10299.
70. Rackis, J. J., H. A. Sasame, R. K. Mann, R. L. Anderson, and A. K. Smith. 1962. Soybean trypsin inhibitors: isolation, purification and physical properties. Arch. Biochem. Biophys. 98:471-478.
71. Banachowicz, E., J. Gapinski, and A. Patkowski. 2000. Solution structure of biopolymers: a new method of constructing a bead model. Biophys. J. 78:70-78.
72. Li, C. H. 1958. Symposium on Protein Structure. Wiley & Sons, New York.
73. Cunningham, L. W., Jr., F. Tietze, N. M. Green, and H. Neurath. 1953. Molecular kinetic properties of trypsin and related proteins. Discuss. Farad. Soc. 13:58-67.
74. Lewis, U. J., D. E. Williams, and N. G. Brink. 1956. Pancreatic elastase: purification, properties, and function. J. Biol. Chem. 222:705-720.
75. Matsubara, H., C. B. Kasper, D. M. Brown, and E. L. Smith. 1965. Subtilisin bpn'. I. Physical properties and amino acid composition. J. Biol. Chem. 240:1125-1130.
76. Armstrong, J. M., D. V. Myers, J. A. Verpoorte, and J. T. Edsall. 1966. Purification and properties of human erythrocyte carbonic anhydrases J. Biol. Chem. 241:5137-5149.
77. Neurath, H., G. R. Cooper, and J. O. Erickson. 1941. The shape of protein molecules. II. Viscosity and diffusion studies of native proteins. J. Biol. Chem. 138:411-436.
78. Edelhoch, H. 1957. The denaturation of pepsin. I. Macromolecular changes. J. Am. Chem. Soc. 79:6100-6109.
79. Lanni, F., and B. R. Ware. 1984. Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery. Biophys. J. 46:97-110.
80. Newman, J., J. E. Estes, L. A. Selden, and L. C. Gershman. 1985. The presence of oligomers at subcritical actin concentrations. Biochemistry. 24:1538-1544.
81. Isemura, T., and S. Fujita. 1957. Physicochemical studies on taka-amylase a. I. Size and shape detemination by the measurement of sedimentation constant, diffusion constant, and viscosity. J. Biochem. (Japan). 44:443-450.
82. Pedersen, K. O. 1945. Ultracentrifugal Studies on Serum and Serum Fractions. Almquist and Wiksell, Uppsala, Sweden.
83. Oncley, J. L., G. Scatchard, and A. Brown. 1947. Physical-chemical characteristics of certain of the proteins of normal human plasma. J. Phys. Colloid Chem. 51:184-198.
84. Charlwood, P. A. 1952. Sedimentation and diffusion of human albumins. 1. Normal human albumins at a low concentration. Biochem. J. 51:113-118.
85. Wood, E., D. Dalgleish, and W. Bannister. 1971. Bovine erythrocite cupro-zinc protein. 2. Physicochemical properties and circular dichroism. Eur. J. Biochem. 18:187-193.
86. Ogston, A. G. 1949. The Gouy diffusiometer; further calibration. Proc. Roy. Soc. (London). 196:272-285.
87. Huet, M., and J.-M. Claverie. 1978. Sedimentation studies of the reversible dimer-tetramer transition kinetics of concanavalin A. Biochemistry. 17:236-241.
88. Sanders, A. H., D. L. Purich, and D. S. Cannell. 1981. Oxygenation of hemoglobin: correspondence of crystal and solution properties using translational diffusion constant measurements. J. Mol. Biol. 147:583-595.
89. Hammerstedt, T. H., H. Möhler, K. A. Decker, and W. A. Wood. 1971. Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase. I. Physical evidence for a three-subunit molecule. J. Biol. Chem. 246:2069-2074.
90. Altman, P. L., and D. S. Dittmer. 1972. Biology Data Book, Vol. I, 2nd ed. FASEB, Bethesda, MD.
91. Wu, J.-Y., and J. T. Yang. 1970. Physicochemical characterization of citrate synthase and its subunits. J. Biol. Chem. 245:212-218.
92. Sumner, J., N. Gralén, and I. Eriksson-Quensel. 1938. The molecular weights of canavalin, concananvalin A, and concanavalin B. J. Biol. Chem. 125:45-48.
93. Cecil, R., and A. G. Ogston. 1948. Addendum: Sedimentation and diffusion of glucose oxidase (notatin). Biochem. J. 42:229.
94. Kusai, K., I. Sekuzu, B. Hagihara, K. Okunuki, S. Yamauchi, and M. Nakai. 1960. Crystallization of glucose oxidase from Penicillium amagasakiense. Biochim. Biophys. Acta. 40:555-557.
95. Glikina, M. V., and P. A. Finogenov. 1950. Investigation of muscular aldolase in various stages of isolation. Biokhimiya. 15:457-464.
96. Kawahara, K. 1969. Evaluation of diffusion coefficients of proteins from sedimentation boundary curves. Biochemistry. 8:2551-2557.
97. Taylor, J. F., A. A. Green, and G. T. Cori. 1948. Crystalline aldolase. J. Biol. Chem. 173:591-604.
98. Christen, P., H. Göschke, F. Leuthardt, and A. Schmid. 1965. Über die aldolase der kaninchenleber molekulargewicht, dissoziation in untereinheiten. Helv. Chim. Acta. 48:1050-1056.
99. Fischer, E. H., D. C. Teller, and V. L. Seery. 1967. A reinvestigation of the molecular weight of glycogen phosphorylase. Biochemistry. 6:3315-3327.
100. Fitori, J. 1971. Dielectric dispersion of phosphorylase b. Acta. Biochim. et Biophys. 6:427-432.
101. Hellweg, T., W. Eimer, E. Krahn, K. Schneider, and A. Müller. 1997. Hydrodynamic properties of nitrogenase. The MoFe protein from azotobacter vinelandii as studied by dynamic light scattering and hydrodynamic modeling. Biochim. Biophys. Acta. 1337:311-318.
102. Sumner, J., and N. Gralén. 1938. The molecular weight of crystalline catalase. Science. 87:284.
103. Samejima, T. 1959. Splitting of the catalase molecule by alkali treatment. J. Biochem. (Tokyo). 46:155-159.
104. Hahn, D. K., and S. R. Aragon. 2006. Intrinsic viscosity of proteins and Platonic solids by boundary element methods. J. Chem. Theory Comput. In press.
105. Beeser, S. A., D. P. Goldenberg, and T. G. Oas. 1997. Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI. J. Mol. Biol. 269:154-164.
106. Spooner, P. J. R. and A. Watts. 1991. Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1. Evidence from deuterium NMR measurements. Biochemistry. 30:3871-3879.
107. Bauer, D. R., S. J. Opella, D. J. Nelson, and R. Pecora. 1975. Depolarized light scattering and carbon nuclear resonance measurements of the isotropic rotational correlation time of muscle calcium binding protein. J. Am. Chem. Soc. 97:2580-2582.
108. Dill, K. and A. Allerhand. 1979. Small errors in C H bond lengths may cause large error in rotational correlation times determined from carbon-13 spin-lattice relaxation measurements. J. Am. Chem. Soc. 101:4376-4378.
109. Aramini, J. A., T. Drakenberg, T. Hiraoki, Y. Ke, K. Nitta, and H. J. Vogel. 1992. Calcium-43 NMR studies of metal ion binding to alpha-lactalbumins and horse and pigeon lysozyme. Biochemistry. 31:6761-6768.
110. Mahoney, N. M., V. K. Rastogi, S. M. Cahill, M. E. Girvin, and S. C. Almo. 2000. Binding orientation of proline-rich peptides in solution: polarity of the profilin-ligand interaction. J. Am. Chem. Soc. 122:7851-7852.
111. Wahl, P., and S. N. Timasheff. 1969. Polarized fluorescence decay curves for β-lactoglobulin A in various states of association. Biochemistry. 8:2945-2949.
112. Miura, N., N. Asaka, N. Shinyashiki, and S. Mashimo. 1994. Microwave dielectric study on bound water of globule proteins in aqueous solution. Biopolymers. 34:357-364.
113. Steiner, R. F. 1954. Reversible association processes of globular proteins. VI. The combination of trypsin with soybean inhibitor. Arch. Biochem. Biophys. 49:71-92.
114. Maliwal, B. P., and J. R. Lackowicz. 1984. Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues. Biophys. Chem. 19:337-344.
115. 15N-NMR relaxation measurements. Eur. J. Biochem. 235:629-640.
116. Kask, P., P. Piksarv, Ü. Mets, M. Pooga, and E. Lippmaa. 1987. Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B. Eur. Biophys. J. 14:257-261.
117. Mihashi, K., and P. Wahl. 1975. Nanosecond pulsefluorometry in polarized light of G-actin-epsilon-ATP and F-actin-epsilon-ADP. FEBS Lett. 52:8-12.
118. Wahl, P. 1966. Détermination du temps de relaxation brownienne de la sérum-albumine en solution par la mesure de la décroissance de la fluorescence polarisée. C. R. Acad. Sci. Paris. 263D:1525-1528.
119. Helms, M. K., C. E. Petersen, N. V. Bhagavan, and D. M. Jameson. 1997. Time-resolved fluorescence studies on site-directed mutants of human serum albumin. FEBS Lett. 408:67-70.
120. Castellano, F. N., J. R. Lakowicz, and J. D. Dattelbaum. 1998. Long-lifetime Ru(II) complexes as labeling reagents for sulfhydryl groups. Anal. Biochem. 255:165-170.
121. Lakowicz, J. R., and I. Gryczynski. 1992. Tryptophan fluorescence intensity and anisotropy decays of human serum albumin resulting from one- and two-photon excitation. Biophys. Chem. 45:1-6.
122. Yang, D. C. H., W. E Gall, and G. M. Edelman. 1974. Rotational correlation time of concanavalin A after interaction with a fluorescent probe. J. Biol. Chem. 249:7018-7023.
123. Johnson, M. E., L. W. -M. Fung, and C. Ho. 1977. Magnetic field and temperature induced line broadening in the hyperfine-shifted proton resonances of myoglobin and hemoglobin. J. Am. Chem. Soc. 99:1245-1250.
124. Schlecht, P., A. Mayer, H. Vogel, and G. Hettner. 1969. Dielectric properties of hemoglobin and myoglobin: influence of particle size and solvent on the dielectric dispersion. Biopolymers.7:963-974.
125. Halle, B., T. Andersson, S. Forsén, and B. Lindman. 1981. Protein hydration from water oxygen-17 magnetic relaxation. J. Am. Chem. Soc. 103:500-508.
126. 2H magnetic relaxation. Biochemistry. 15:4255-4264.
127. Anderson, S. R. 1969. Fluorescence polarization studies of conjugates of beef heart lactic dehydrogenase with 1-dimethylaminonaphthalene-5-sulfonyl chloride. Biochemistry. 8:1394-1396.
128. Chang, Y.-C., R. D. Scott, and D. J. Graves. 1986. Function of pyridoxal-5′-phosphate in glycogen phosphorylase: F-19 NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate. Biochemistry. 25:1932-1939.
129. Tung, M. S., and R. F. Steiner. 1975. The use of nanosecond fluorometry in detecting conformational transitions of an allosteric enzyme. Biopolymers. 14:1933-1949.
130. Aragon, S. R., and D. K. Hahn. 2005. The polarizability and capacitance of Platonic solids and the Kerr constant of proteins. Lecture Series in Computer and Computational Sciences. Brill, Leiden. 4:25-32.
131. Anderson, E., Z. Bai, C. Bischof, S. Blackford, J. Demmel, J. Dongarra, J. Du Croz, A. Greenbaum, S. Hammarling, A. McKenney, and D. Sorensen. LAPACK User's Guide, 3rd ed., SIAM, Philadelphia, 1999.