Measurement of the Force - Velocity Relation for Growing Microtubules

8. S.-J. Lin, R. A. Pufahl, A. Dancis, T. V. O’Halloran, V. Cizewski Culotta, J. Biol. Chem. 272, 9214 (1997). 9. L. W. J. Klomp et al., ibid., p. 9221. 10. C. Vulpe et al., Nature Genet. 3, 7 (1993). 11. P. C. Bull et al., ibid. 5, 327 (1993); R. E. Tanzi et al., ibid., p. 344. 12. S. Lutsenko and J. H. Kaplan, Biochemistry 34, 15607 (1995). 13. M. J. Petris et al., EMBO J. 15, 6084 (1996). 14. P. C. Bull and D. W. Cox, Trends Genet. 10, 246 (1994); P.-O. Eriksson and L. Sahlman, J. Biomol. NMR 3, 613 (1993); J. L. Hobman and N. L. Brown, in Metal Ions in Biological Systems, A. Sigel and H. Sigel, Eds. (Dekker, New York, 1997), vol. 34, pp. 527–568. 15. ATX1 was cloned into pET11d (Novagen) and expressed in E. coli strain BL21(DE3) after induction with IPTG. The protein was isolated by a freeze-thaw extraction and purified to homogeneity by DEAESephacel batch treatment and subsequent chromatography on CM Sepharose FF (Pharmacia). Approximately 6 to 10 mg of pure protein was obtained per liter of bacterial culture. Protein concentration was determined from absorbance at 280 nm (with an extinction coefficient of 4400 M21 cm21, calculated on the basis of total amino acid composition) or by Bradford assay [M. Bradford Anal. Biochem. 72, 248 (1976)] with immunoglobulin G as a standard. Electrospray mass spectrometry (ES-MS) of apo-Atx1 revealed a single peak of 8088.1 daltons, which corresponds to full-length Atx1 lacking its NH2-terminal methionine; in addition, ES-MS of Hg-Atx1 yielded one peak at 8287.6 daltons. Gel filtration on an HPLC QC-PAK TSK 200GL ( TosoHaas) column yielded molecular sizes of 10,200, 9840, and 9960 daltons for apo-, Hg(II)-, and Cu(I)-Atx1, respectively. 16. Cu(I)-Atx1 was typically prepared by incubation of apoprotein with a Cu(II) salt in the presence of a thiol reductant. All manipulations were performed in an inert atmosphere at 4°C. CuSO4 (2.5 equivalents) was added, with stirring, to a solution of apo-Atx1 and DTT (20-fold molar excess relative to protein) in 50 mM tris (pH 8.0, adjusted with solid MES to avoid Cl2). Excess metal was removed by ultrafiltration and the protein was exchanged into a solution containing 20 mM MES (pH 6.0) and 20% (v/v) glycerol. The final protein concentration was 7.84 mM with a Cu/protein ratio of 0.75 6 0.02. Solutions (.2 mM) of the Cu(I)-protein can be handled in air at 4°C for 30 min or stored long term (.4 months) at 270°C with no observable formation of Cu(II). 17. XAS data were obtained at Stanford Synchrotron Radiation Laboratory (SSRL) beam line VII-3 as fluorescence excitation spectra with a Ge solid-state detector array. Samples (150 ml) were maintained at 10 K throughout measurements. Data reduction and analysis were performed according to standard procedures [P. J. Riggs-Gelasco, R. Mei, C. F. Yocum, J. E. Penner-Hahn, J. Am. Chem. Soc. 118, 2387 (1996)]. Data were fitted with amplitude and phase functions calculated with FEFF 6.01 [J. J. Rehr et al., ibid. 113, 5135 (1991)], calibrated by fitting model compounds of known structure. 18. L.-S. Kau et al., ibid. 109, 6433 (1987). 19. D. R. Winge et al., J. Bioinorg. Chem. 2, 2 (1997). 20. E. I. Solomon et al., Chem. Rev. 96, 2563 (1996). 21. H. H. Thorp, Inorg. Chem. 31, 1585 (1992). 22. I. J. Pickering et al., J. Am. Chem. Soc. 115, 9498 (1993). 23. D. Coucouvanis et al., Inorg. Chem. 19, 2993 (1980). 24. S. A. Koch et al., ibid. 23, 122 (1984). 25. 199Hg-Atx1 was prepared by the addition of 199Hg(II) to apo-Atx1 in 20 mM MES (pH 6.0). DT T was removed from apo-Atx1 by ultrafiltration before the addition of mercury. 199Hg(II) (1.2 equivalents) was added to apoAtx1 on ice with stirring. Free metal was removed with several successive washes with buffer by ultrafiltration followed by exchange into D2O buffer. The final protein concentration was 2.40 mM with a 199Hg/protein ratio of 0.83 6 0.05 in a solution containing 25 mM sodium phosphate (pH* 6.6), 50 mM NaCl, and 98% D2O. A similar protocol was used to prepare the EXAFS sample except that Cl2 was not present in any of the buffers. The final protein concentration was 3.36 mM with a Hg/protein ratio of 0.75 6 0.06 in 20 mM MES (pH 6.0). 26. J. G. Wright et al., Prog. Inorg. Chem. 38, 323 (1990). 27. L. M. Utschig et al., Science 268, 380 (1995). 28. L. M. Utschig et al., Inorg. Chem. 36, 2926 (1997). 29. The 1H{199Hg} HMQC spectra of 199Hg-Atx1 were obtained on a Bruker 600-MHz spectrometer (14.09 T, 107.4 MHz for 199Hg) equipped with a 5-mm tunable probe. The pulse sequence and optimal delays for Met, Cys, and His ligands have been described (27). Spectral widths of 7246 Hz for 1H (F2) and 32,221 Hz for 199Hg (F1) were used. A total of 64 t1 blocks was accumulated, with 1024 transients collected in F2 per block. The 1H 90° pulse was 8.0 ms and the 199Hg(90°)21 value was 20.4 ms, as calibrated with Hg(CH3)2. Safety note: The latter compound is permeable to latex gloves and is exceedingly toxic. Discussion of alternative compounds can be found at www.chem.nwu.edu/;ohallo/HgNMRStandards 30. G. R. Dieckmann et al., J. Am. Chem. Soc. 119, 6195 (1997). 31. J. R. R. Frausto da Silva and R. J. P. Williams, in The Biological Chemistry of the Elements (Clarendon, Oxford, 1991), pp. 396–397. 32. B. V. Cheesman, A. P. Arnold, D. L. Rabenstein, J. Am. Chem. Soc. 110, 6359 (1988). 33. The two-hybrid analysis [L. Guarente, Proc. Natl. Acad. Sci. U.S.A. 90, 1639 (1993)] was performed with the MATCHMAKER System (Clontech). Nucleotides 11 to 1222 of ATX1 were amplified by PCR and inserted at the Bam HI and Pst I sites of the pGBT9 plasmid encoding the DNA-binding domain of Gal4, to yield pPS002. For preparation of vector pPS001, CCC2 nucleotides (nt) 11 to 1745 were amplified and inserted at the Eco RI and Sal I sites of pGAD424 encoding the Gal4 activation domain. The remaining three Ccc2-Gal4 fusion constructs were obtained by amplifying the corresponding CCC2 sequences and inserting them into the Bam HI and Pst I sites of pGAD424. Plasmid pCF12 contains CCC2 nt 11163 to 11582, pCF34 contains nt 11731 to 12711, and pCF56 contains nt 12831 to 13294. Yeast strain SFY526 was cotransformed with the vectors based on pGBT9 and pGAD424, and grown in 5 ml of synthetic dextrose (SD) medium to an optical density at 600 nm of 4.0. Cells were harvested, washed in water, and resuspended in 1 ml of 50 mM sodium phosphate buffer (pH 7.0) containing 10 mM KCl, 1 mM MgSO4, and 40 mM b-mercaptoethanol. They were lysed by homogenization with glass beads after adding 45 ml of 0.1% SDS. The resulting extract (800 ml) was mixed with 200 ml of o-nitrophenyl-b-D-galactopyranoside (4 mg/ml) and incubated at 30°C overnight. After centrifugation, b-Gal activity was measured in the supernatant at 420 nm. In some instances, cells were grown in the presence of the Cu chelator BCS (3 mM). Results are representative of two to four independent samples with ranges of #15%. b-Gal activity units for the p53/SV40 positive controls (and vectors) were 9.75 (0.21) in the absence of BCS and 9.95 (0.27) in the presence of BCS. 34. R. P. Hausinger et al., Eds., Mechanisms of Metallocenter Assembly ( VCH, New York, 1996). 35. R. A. Steele and S. J. Opella, Biochemistry 36, 6885 (1997). 36. J. Gitschier and W. Fairbrother, personal communication. 37. V. Cizweski Culotta et al., J. Biol. Chem. 272, 23469 (1997 ). 38. Supported by NIH: GM-54111 ( T.V.O.), GM-38047 (J.E.P.-H.), GM-50016 ( V.C.C.), Biophysics Training Grant T32GM08382 (C.P.S.), Training Grant ES 07141 (P.J.S.), and F32 DK-09305 (R.A.P.). SSRL is funded by the Department of Energy (Office of Basic Energy Sciences and Office of Health and Environmental Research) and by the NIH Biomedical Research Technology Program. The Northwestern University 600-MHz NMR Facility is funded by the W. M. Keck Foundation, NSF, NIH, and the R. H. Lurie Cancer Center. We thank R. Scott for the Cu(SC6H5)3 22 XANES data, D. Huffman for [Cu(II)(imidazole)4](NO3)2, and A. Duncan for the color illustration.