RNA and Protein Complexes of trp RNA-Binding Attenuation Protein

Anal. Chem. 2009, 81, 2218–2226

RNA and Protein Complexes of trp RNA-Binding Attenuation Protein Characterized by Mass Spectrometry Satoko Akashi,* Masahiro Watanabe, Jonathan G. Heddle,† Satoru Unzai, Sam-Yong Park, and Jeremy R. H. Tame* Yokohama City University, Supramolecular Biology, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus, and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a “snapshot” of all the species in solution. In living cells, biological events are regulated by various macromolecules interacting with each other, often creating very large complexes. Precise mass measurements can greatly aid understanding of these complexes, even when structural information is available. Advances in electrospray ionization mass spectrometry (ESI-MS) have recently enabled observation of intact ions of giant noncovalent biological macromolecular complexes including the E. coli ribosome,1 GroEL,2-4 20S proteasome,5-7 and RNA polymerase.8 These studies have demonstrated that ESI-MS provides complementary information to that provided by conventional structural biology techniques, such as X-ray crystallography, NMR, and electron microscopy, and that it offers significant * To whom correspondence should be addressed. E-mail: akashi@ tsurumi.yokohama-cu.ac.jp (S.A.); [email protected] (J.R.H.T.). Phone: +81-45-508-7217 (S.A.); +81-45-508-7228 (J.R.H.T.). Fax: +81-45-508-7362 (S.A.); +81-45-508-7366 (J.R.H.T.). † Present address: Global Edge Institute, Tokyo Institute of Technology, 4259, S2-17, Nagatsuda, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.

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insight into the biophysical properties of complexes which play critical roles in various processes. In the present study, we have used ESI-MS to characterize trp RNA-binding attenuation protein (TRAP) and its complexes with RNA or its regulator protein. The transcription and translation of the trp operon in Bacillus is highly regulated to maintain an appropriate level of intracellular free tryptophan (Trp), and this operon has yielded a wealth of information regarding various methods of gene regulation.9-13 If sufficient Trp is present in the cytoplasm, it binds to TRAP, enabling the protein to bind a specific RNA sequence found in the upstream region of trp mRNA. This triggers the formation of a termination loop so that the coding region for tryptophan synthetic enzymes is not transcribed. If the level of charged tRNATrp falls, anti-TRAP protein (AT) binds to Trp-activated TRAP and prevents its interaction with RNA.14,15 TRAP has been the subject of numerous biological and biophysical studies, and crystal structures are available for TRAP complexed with Trp,16,17 with Trp and RNA,18 and with Trp and AT.19 TRAP from the mesophile B. subtilis and the thermophile B. stearothermophilus show 77% sequence identity and form highly similar ring structures composed of 11 monomers, giving a total molecular (1) Rostom, A. A.; Fucini, P.; Benjamin, D. R.; Juenemann, R.; Nierhaus, K. H.; Hartl, F. U.; Dobson, C. M.; Robinson, C. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5185–5190. (2) Rostom, A. A.; Robinson, C. V. J. Am. Chem. Soc. 1999, 121, 4718–4719. (3) van Duijn, E.; Bakkes, P. J.; Heeren, R. M.; van den Heuvel, R. H.; van Heerikhuizen, H.; van der Vies, S. M.; Heck, A. J. Nat. Methods 2005, 2, 371–376. (4) van Duijn, E.; Simmons, D. A.; van den Heuvel, R. H.; Bakkes, P. J.; van Heerikhuizen, H.; Heeren, R. M.; Robinson, C. V.; van der Vies, S. M.; Heck, A. J. J. Am. Chem. Soc. 2006, 128, 4694–4702. (5) Chernushevich, I. V.; Thomson, B. A. Anal. Chem. 2004, 76, 1754–1760. (6) Sharon, M.; Witt, S.; Felderer, K.; Rockel, B.; Baumeister, W.; Robinson, C. V. J. Biol. Chem. 2006, 281, 9569–9575. (7) Loo, J. A.; Berhane, B.; Kaddis, C. S.; Wooding, K. M.; Xie, Y.; Kaufman, S. L.; Chernushevich, I. V. J. Am. Soc. Mass Spectrom. 2005, 16, 998– 1008. (8) Lorenzen, K.; Vannini, A.; Cramer, P.; Heck, A. J. Structure 2007, 15, 1237– 1245. (9) Babitzke, P. Curr. Opin. Microbiol. 2004, 7, 132–139. (10) Babitzke, P.; Stults, J. T.; Shire, S. J.; Yanofsky, C. J. Biol. Chem. 1994, 269, 16597–16604. (11) Babitzke, P.; Bear, D. G.; Yanofsky, C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7916–7920. (12) Babitzke, P. Mol. Microbiol. 1997, 26, 1–9. (13) Gollnick, P.; Babitzke, P.; Antson, A.; Yanofsky, C. Annu. Rev. Genet. 2005, 39, 47–68. (14) Valbuzzi, A.; Yanofsky, C. Science 2001, 293, 2057–2059. (15) Valbuzzi, A.; Gollnick, P.; Babitzke, P.; Yanofsky, C. J. Biol. Chem. 2002, 277, 10608–10613. 10.1021/ac802354j CCC: $40.75  2009 American Chemical Society Published on Web 02/16/2009

mass of around 92 kDa.16,17 Mass spectrometry studies provided the first hint that a small population of 12mer rings may also exist in solution.20 We have since shown that by linking several TRAP monomers together with peptide linkers it is possible to create artificial 12mer TRAP rings.21,22 Crystal structures of these 12mers show the contacts between subunits are almost completely unchanged from the native 11mer form. Crystallography suggests that the 12mer TRAP ring binds to both Trp and AT in the same mode as the 11mer, although the complex formed from 11mer TRAP and AT proved uncrystallizable.19 This is because the 12mer TRAP-AT complex is selectively crystallized due to the molecular point group symmetry being particularly amenable to the formation of crystal contacts. Even wild-type TRAP therefore crystallizes to give a 12mer ring in the presence of AT. We have used ESI-MS to characterize the complexes formed by wild-type TRAP and mutant 12mer TRAP with RNA or AT. The previous mass spectrometry studies of B. subtilis TRAP, carried out by McCammon et al., showed not only single- but also double- and triple-ring arrangements of TRAP under mild conditions, in the presence of a 4-fold excess of Trp per monomer of TRAP in 20 mM ammonium acetate.20 Under harsher desolvation conditions with increasing acceleration of ions in the source, two species, 11mer TRAP and 12mer TRAP, were separately observed in the mass spectrum. MS/MS experiments of the single- and double-ring arrangements of TRAP were also carried out to characterize TRAP in the gas phase. Ruotolo et al. reported ESI-MS of the B. subtilis TRAP bound with Trp and RNA, but their discussion focused on the preservation of the ring shape of TRAP in the gas phase based on experiments using an ion mobility mass spectrometer.23 Even though complexes formed by TRAP are central to the mechanism of regulation of Trp synthesis, little ESI-MS has been carried out to study TRAP binding to its target macromolecules, especially AT. Here we report ESI-MS for wild-type and mutant TRAP and their complexes and compare the results with those from X-ray crystallography and other analytical methods.19 EXPERIMENTAL SECTION Two species of TRAP were studied, wild-type TRAP from B. stearothermophilus and an artificial mutant created by linking three copies of B. stearothermophilus TRAP with linkers of seven alanine residues. The production and crystal structure of this protein have been described previously.21,22 Throughout this manuscript, the two TRAP proteins used are referred to as “wild-type” and “T3A7”, respectively. Preparation of Wild-Type TRAP and T3A7. Wild-type TRAP and mutant T3A7 were prepared as previously reported.19 Briefly, (16) Antson, A. A.; Otridge, J.; Brzozowski, A. M.; Dodson, E. J.; Dodson, G. G.; Wilson, K. S.; Smith, T. M.; Yang, M.; Kurecki, T.; Gollnick, P. Nature 1995, 374, 693–700. (17) Chen, X.; Antson, A. A.; Yang, M.; Li, P.; Baumann, C.; Dodson, E. J.; Dodson, G. G.; Gollnick, P. J. Mol. Biol. 1999, 289, 1003–1016. (18) Antson, A. A.; Dodson, E. J.; Dodson, G.; Greaves, R. B.; Chen, X.; Gollnick, P. Nature 1999, 401, 235–242. (19) Watanabe, M.; Heddle, J. G.; Kikuchi, K.; Unzai, S.; Akashi, S.; Park, S. Y.; Tame, J. R. H. Proc. Natl. Acad. Sci. U.S.A., in press. (20) McCammon, M. G.; Herna´ndez, H.; Sobott, F.; Robinson, C. V. J. Am. Chem. Soc. 2004, 126, 5950–5951. (21) Heddle, J. G.; Yokoyama, T.; Yamashita, I.; Park, S. Y.; Tame, J. R. Structure 2006, 14, 925–933. (22) Watanabe, M.; Mishima, Y.; Yamashita, I.; Park, S. Y.; Tame, J. R.; Heddle, J. G. Protein Sci. 2008, 17, 518–526. (23) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658–1661.

wild-type TRAP and T3A7 were expressed in E. coli BL21(DE3) cells (Strategene). The harvested cells were resuspended in a buffer consisting of 50 mM Tris-HCl (pH 8.5), 100 mM NaCl, lysed by sonication, and centrifuged at 19 000 rpm at 4 °C for 30 min. The supernatant was applied to a HiTrap Q Sepharose column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 8.5), and the protein was eluted using an NaCl gradient. TRAP- or T3A7-containing fractions were collected and exchanged into 20 mM MES (pH 6.5), 100 mM NaCl, and applied to a HiTrap Heparin Sepharose column (GE Healthcare) equilibrated in the same buffer. Protein was eluted using an increasing NaCl gradient. TRAP- or T3A7-containing fractions were collected and dialysed against 50 mM Tris-HCl (pH 8.5), 300 mM NaCl, and concentrated using an Amicon ultracentrifugal filter (Millipore). Concentrated samples were applied to a Superdex 200 column (GE Healthcare) equilibrated in the same buffer, and sample fractions of wild-type TRAP or T3A7 were collected. Preparation of AT. AT protein was prepared as previously reported.19 Briefly, B. subtilis AT was expressed in E. coli BL21(DE3) cells (Strategene). The harvested cells were resuspended in a buffer consisting of 50 mM Tris-HCl (pH 8.5), 1 mM DTT, lysed by sonication, and centrifuged at 19 000 rpm at 4 °C for 30 min. The supernatant was heated at 85 °C for 10 min. AT remained soluble after heat treatment. Precipitant was removed by centrifugation (15 000 rpm at 4 °C for 30 min), and supernatant was applied directly to a HiTrap Q Sepharose column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 8.5), 1 mM DTT and was eluted using an increasing NaCl gradient. Ammonium sulfate was added to the pooled AT fractions to a final concentration of 1 M before the sample was applied to a Phenyl Sepharose column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 8.5), 1 M ammonium sulfate, 1 mM DTT. The protein was eluted with a decreasing ammonium sulfate gradient and was found to elute between 0.8 and 0.3 M ammonium sulfate. The pooled protein was concentrated using a Centriprep centrifugal filter device (Millipore). After concentration it was applied to a Superdex 200 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1 mM DTT and purified. Sample Preparation for NanoESI mass Spectrometry (NanoESI-MS). For nanoESI-MS of wild-type TRAP and T3A7, sample solutions with a concentration of 10 µM (for 11mer or 12mer) were prepared by extensive dialysis of TRAP solutions in a buffer consisting of 50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1 mM DTT against 50 mM ammonium acetate. For the analysis of the TRAP-RNA complex, 55mer single-stranded RNA consisting of 11 UAGCC repeats (Dharmacon) was used. TRAP solutions in a buffer consisting of 50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1 mM DTT, 0.5 mM Trp were dialyzed against 50 mM ammonium acetate and then mixed with RNA solution in 50 mM ammonium acetate at a molar ratio of wild-type TRAP/RNA ) 1:1 or T3A7/RNA ) 1:0.25. For nanoESI-MS of the TRAP-AT complexes, TRAP solutions in a buffer consisting of 50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1 mM DTT, 0.5 mM Trp and AT solutions in a buffer consisting of 50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 1 mM DTT were first dialyzed against 500 mM or 1 M ammonium acetate, then mixed at a molar ratio of TRAP (or T3A7)/AT ) 1:6 (TRAP 11mer or T3A7 12mer/AT trimer). The final concentration of TRAP Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Figure 1. NanoESI mass spectra of wild-type TRAP and T3A7 in 50 mM ammonium acetate. For wild-type TRAP, the spectra were obtained with cone and collision voltages of (a) 100 V/50 V, (b) 120 V/50 V, and (c) 150 V/50 V, respectively. For T3A7, the spectra were obtained with cone and collision voltages of (d) 120 V/40 V, and (e) 150 V/70 V, respectively. The numbers of monomer subunits in each TRAP protein are indicated at the peaks in the mass spectra. Insets are expanded mass spectra in the mass range of m/z 4460-4600 for wild-type TRAP and those of m/z 4630-4770 for T3A7. Open circles represent satellite peaks for 11mer or 12mer TRAP associated with Trp residues. A peak labeled with an asterisk corresponds to the species for which the definite number of subunits cannot be determined from the m/z value.

11mer or T3A7 12mer in the TRAP-AT solution was 10 µM for nanoESI-MS. NanoESI Mass Spectrometry. NanoESI mass spectra were acquired by Q-Tof2 (Waters) with a nanoESI ion source. The mass spectra were calibrated with (CsI)nCs+ up to m/z 8000 or 10 000. MassLynx version 3.5 software (Waters) was used for data processing and peak integration. The parameters for ESI mass spectrometry were similar to those previously reported.24 The temperature of the ion source was set at 80 °C. An aliquot of 4 µL of sample solution was deposited in a nanospray glass tip (Waters) and introduced into a nanoESI source. To observe high m/z ions, the pressure in the quadrupole ion guide of the Q-Tof2 was maintained at 8 × 10-3 Pa by throttling down the Speedivalve fitted to the rotary pump for the ion source region. Cone and collision voltages were optimized for each measurement. Each mass spectrum from m/z 2000 or m/z 2500 to m/z 8000 or m/z 10 000 was acquired in 4 s, and several spectra were accumulated and smoothed by the Savitzky-Golay (24) Itoh, Y.; Unzai, S.; Sato, M.; Nagadoi, A.; Okuda, M.; Nishimura, Y.; Akashi, S. Proteins 2005, 61, 633–641.

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method. Centroid-top (80%) m/z values were used for calculation of molecular masses. RESULTS NanoESI Mass Spectra of Wild-Type TRAP and T3A7. Purified recombinant wild-type TRAP and T3A7 were extensively dialyzed against 50 mM ammonium acetate and subjected to nanoESI-MS. Figure 1 shows the ESI mass spectra obtained at various cone and collision voltages. In the case of wild-type TRAP, the peaks observed in the mass spectra changed according to the cone and collision voltages. Figure 1a shows the spectrum obtained at cone and collision voltages of 100 and 50 V, respectively. Intense signals at m/z 3900-4600 correspond to 23+, 22+, 21+, and 20+ ions of TRAP 11mer ring, showing the molecular mass to be 90 712.1 ± 9.4, which was calculated using the m/z value of each 11mer peak. The theoretical mass for TRAP 11mer ring without Trp is 90 666.1, whereas that for Trp-saturated TRAP 11mer is 92 912.6. The observed mass value suggests that not every TRAP subunit was associated with a Trp molecule, and satellite peaks suggesting the attachment of 1-4 Trp molecules to the TRAP 11mer ring were also found, as shown in the inset of

Table 1. Observed and Theoretical masses of the Wild-Type TRAP and T3A7 obsd mass (charge number)a

exptl mass of the molecule (Da)a

theor mass without Trp (Da)

theor mass with Trp (Da)

wild-type TRAP 11mer

4122.9 (22+)b 4320.0 (21+) 4535.7 (20+)

90 694.4 ± 3.3

90 666.1

92 912.6


4498.3 (22+) 4712.2 (21+) 4946.7 (20+)

98 936.5 ± 4.3

98 908.5

101 359.2


4581.6 (18+)b 4850.9 (17+) 5153.9 (16+) 5497.6 (15+) 5890.0 (14+) 6342.9 (13+)

82 448.7 ± 2.1

82 423.7

84 4666.0

wild-type TRAP 9mer

5709.3 (13+) 6184.5 (12+) 6747.0 (11+) 7421.5 (10+)

74 205.1 ± 1.9

74 181.3

76 019.4

wild-type TRAP 8mer

6597.4 (10+) 7330.0 (9+)

65 963.1 ± 1.6

65 939.0

67 572.8

wild-type TRAP 6mer

3091.8 (16+) 3297.9 (15+)b 3533.2 (14+)

49 453.6 ± 0.3

49 454.2

50 679.6

wild-type TRAP 5mer

3747.7 (11+) 4122.9 (10+)b 4581.6 (9+)b

41 218.6 ± 5.5

41 211.9

42 233.0

wild-type TRAP 4mer

3297.9 (10+)b 3664.0 (9+) 4122.9 (8+)b

32 969.4 ± 1.2

32 969.5

33 786.4

T3A7 12mer

4112.6 (25+) 4283.2 (24+) 4470.1 (23+) 4672.7 (22+) 4895.0 (21+) 5140.2 (20+)

102 780.5 ± 6.4

102 744.8

105 195.5

T3A7 9mer

5506.5 (14+) 5930.8 (13+) 6424.5 (12+) 7008.3 (11+) 7709.4 (10+)

77 082.0 ± 3.6

77 058.6

78 896.6

T3A7 6mer

3427.0 (15+)b 3670.4 (14+) 3952.9 (13+)

51 374.1 ± 0.6

51 372.4

52 597.7

T3A7 3mer

2569.6 (10+) 2855.0 (9+) 3211.6 (8+)b 3670.5 (7+)

25 685.9 ± 0.6

25 686.2

26 298.9

protein

b

a “obsd mass” corresponds to the most intense peak for each charge state and was used for the calculation of “exptl mass of the molecule”. Peaks might be overlapped with multiply charged molecular ions of other multimers.

Figure 1a. The molecular mass difference between these satellite peaks was 207-213 Da, which corresponds to a Trp molecule. A weak 22+ peak of the TRAP 12mer ring, which was associated with satellite peaks originated from Trp binding, could also be recognized around m/z 4500. This implies that wild-type TRAP favors 11mer ring formation but there is a minor population of 12mer ring, as observed in the previous study of B. subtilis TRAP.20 When the cone voltage was increased to 120 V, keeping the collision voltage at 50 V (Figure 1b), several new peaks appeared at higher m/z values. 17+, 16+, 15+, 14+, and 13+ ions of TRAP 10mer, and 12+, 11+, and 10+ ions of TRAP 9mer were observed. Protein complexes with similar structure but lower molecular masses are generally observed at lower cone voltage than those with high molecular masses, but TRAP 10mer and 9mer only

appeared at higher cone voltage, as expected for dissociation products of TRAP 11mer. As the cone voltage was increased, the intensity of Trp adducts of TRAP 11mer decreased, as shown in the inset of Figure 1b, implying that Trp residues were released from the TRAP 11mer ring. In addition, the peak width for the 10mer and 9mer obviously narrowed, and the relative intensity of satellite peaks for 10mer and 9mer TRAP was lower than that for TRAP 11mer, suggesting that the 10mer and 9mer do not stably retain Trp residues. Increasing the cone voltage up to 150 V caused considerable dissociation of TRAP 11mer. Masses of the observed peaks in Figure 1c are summarized in Table 1 with the theoretical molecular masses. Peaks of TRAP 10mer (82 448.7 ± 2.1), 9mer (74 205.1 ± 1.9), 8mer (65 963.1 ± 1.6), 6mer (49 453.6 ± 0.3), 5mer (41 218.6 ± 5.5), and 4mer (32 969.4 ± 1.2) were Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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recognized in addition to those of TRAP 11mer and 12mer. Peaks of TRAP 11mer with the molecular mass of 90 694.4 ± 3.3 became narrower than those in the mass spectra at lower cone voltages, and peaks of TRAP 12mer (98 936.5 ± 4.3) could clearly be recognized at m/z 4498.3 (22+), 4712.2 (21+), and 4946.7 (20+), as shown in Figure 1c. When the cone voltage was set at 150 V, most of Trp residues were released from the TRAP 11mer ring, as indicated in the inset of Figure 1c. In contrast to wild-type, the ESI mass spectra of T3A7 showed that it forms a stable 12mer structure, as shown in Figure 1, parts d and e. At lower cone and collision voltages, T3A7 showed intense signals of 12mer protein in the spectrum (Figure 1d). Peaks at m/z 4100-5200 are assigned to 25+, 24+, 23+, 22+, 21+, and 20+ ions of TRAP 12mer ring, corresponding to a molecular mass of 102 802.1 ± 6.6, which was calculated using the m/z value of each 12mer peak. The theoretical mass for the unliganded T3A7 12mer is 102 744.8, and that for the Trp-saturated form is 105 195.5. The observed mass value of T3A7 suggests that not every TRAP subunit was associated with a Trp molecule. As shown in the inset of Figure 1d, the observed peaks for the TRAP 12mer ring of T3A7 were broadened and might not be resolved from Trp-bound TRAP peaks. When the cone and collision voltages were increased to 150 and 70 V, respectively, peaks corresponding to the 3mer (25 685.9 ± 0.6), 6mer (51 374.1 ± 0.6), and 9mer (77 082.0 ± 3.6) proteins were also observed, though the intense signals of 12mer TRAP (102 780.5 ± 6.4) remained, as shown in Figure 1e. Peaks of the Trp-bound satellite peaks could be recognized at higher cone and collision voltages, as shown in the inset of Figure 1e. The masses of the observed peaks in Figure 1e for TRAP multimers are summarized in Table 1 with the theoretical molecular masses. Since T3A7 is a recombinant protein composed of three TRAP monomers covalently bound by two peptide linkers, no other multimers, such as 11mer, 10mer, and so on, were observed. The 3mer, 6mer, and 9mer correspond to one, two, or three polypeptide chains, respectively. At high cone and collision voltages, only slight dissociation of the mutant 12mer TRAP into 3mer, 6mer, and 9mer was observed, implying that the artificial 12mer structure is more stable in the gas phase than the wild-type 11mer. Complex Formation with RNA. Binding of wild-type TRAP and T3A7 to RNA was also examined by ESI-MS. Our previous gel-shift assay revealed that both proteins bind to a single-stranded RNA 55 bases long with a suitable sequence consisting of 11 repeats,22 known to bind strongly to B. subtilis TRAP.25 Although the wild-type showed clear band-shift by complex formation with RNA, T3A7 appeared to form very high molecular weight complexes or precipitate when mixed with RNA, suggesting that binding stoichiometry might be different from that of wild-type or that RNA was binding to multiple TRAP rings.22 Figure 2 shows nanoESI mass spectra of the wild-type and T3A7 mixed with a similar RNA substrate to that used previously.22 In the case of wild-type, the sample solution was prepared by mixing solutions of wild-type TRAP (11mer) and RNA at a molar ratio of 1:1, with both species at a final concentration of 10 µM. As shown in Figure 2a, broad peaks suggesting the molecular mass of 110 638.0 ± 38.5 were observed in the region of m/z 4200-5400. The observed mass (25) Babitzke, P.; Yealy, J.; Campanelli, D. J. Bacteriol. 1996, 178, 5159–5163.

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Figure 2. NanoESI mass spectra of TRAP-RNA complexes (wildtype TRAP (a) and mutant T3A7 (b)) in 50 mM ammonium acetate. For wild-type TRAP and RNA complex, cone and collision voltages were set at 70 and 30 V, respectively, whereas those for T3A7-RNA complex were set at 130 and 25 V.

was approximately 2500 Da larger than the total theoretical mass (108 104.6) of TRAP 11mer with no bound Trp (90 666.1) and RNA (17 438.5) but about 300 Da larger than the theoretical mass (110 351.1) of a 1:1 complex of TRAP 11mer with 11 Trp and RNA. This implies that, as expected, each TRAP monomer has a Trp ligand in the TRAP-RNA complex. Other peaks suggesting the formation of double and triple rings were also observed in the region of m/z 5800-8000, although no such structure was found for wild-type TRAP and T3A7 in the absence of RNA, as shown in Figure 1. Both TRAP-RNA complexes tested were difficult to spray from nanoESI tips because of sample aggregation, completely consistent with the results from gel-shift assays.22 This was more pronounced for T3A7 so the signal intensity of this complex was much lower than that of wild-type TRAP and RNA. When a mixture of 10 µM T3A7 (12mer) and RNA (at a molar ratio of 1:1) was subjected to nanoESI, the nanoESI tip blocked completely and no mass spectrum could be obtained. Figure 2b shows the mass spectrum of 10 µM T3A7 mixed with 2.5 µM RNA. Broad peaks showing the formation of a 1:1 complex of TRAP 12mer and RNA with molecular mass of 122 517.5 ± 27.4 could be observed. Since the theoretical mass of the 1:1 complex of mutant TRAP 12mer and RNA is 120 183.3 without Trp molecules, and 122 634.0 with 12 Trp molecules, it seems most of the binding sites for Trp were apparently occupied. No peaks corresponding to free T3A7 could be observed, even though the protein was present in excess. The quality of mass spectrum for T3A7-RNA was very poor compared to that for wild-type TRAP-RNA complex due to the aggregation of T3A7 upon addition of RNA, leaving only a low concentration in solution. The lower resolution of the spectrum for the T3A7-RNA complex was probably also caused by weakly associated solvent adducts, such as water or ammonium acetate molecules, due to the relatively low collision voltage required to obtain spectra with this sample. Complex Formation with AT. The crystal structure of TRAP-AT complex has recently been solved, showing that the interface between the binding partners is largely hydrophobic,19 so sample solutions for nanoESI-MS were prepared in a relatively high concentration of ammonium acetate, such as 500

Figure 3. NanoESI mass spectrum of 10 µM AT trimer in 500 mM ammonium acetate. The mass spectrum was obtained with cone and collision voltages of 70 and 30 V, respectively. After processing the nanoESI mass spectrum using MassLynx ver. 3.5, the experimental molecular mass for AT multimer was obtained as 68 579.6 ( 1.0, in good agreement with the theoretical mass with 12 zinc ions.

mM or 1 M, in order to stabilize the complex. Figure 3 shows the nanoESI mass spectrum of 10 µM AT trimer in 500 mM ammonium acetate. The concentration of AT was determined from the UV absorbance at 280 nm. In the case of AT alone, however, no drastic difference was found between the spectra of AT in 50 or 500 mM ammonium acetate. Peaks were observed at m/z 3809.7, 4033.7, 4285.6, and 4571.5 corresponding to 18+, 17+, 16+, and 15+ states of AT 12mer, with molecular mass 68 579.6 ± 1.0. The theoretical molecular mass of the AT monomer is 5714.99 with one zinc ion; thus, the observed mass was within error exactly the same as the theoretical mass of AT 12mer with 12 Zn2+, 68 579.9. The crystal structure of AT shows the 12mer is formed from a tetrahedral arrangement of four copies of AT trimer,26 entirely consistent with the present results by nanoESI-MS. Figure 4 shows nanoESI mass spectra of wild-type and mutant TRAP-AT complexes and free AT in 500 mM ammonium acetate. Previous studies of the TRAP-AT complexes by analytical ultracentrifugation showed that binding between TRAP and AT is relatively weak, compared to binding between neighboring TRAP subunits.19 When wild-type TRAP was mixed with AT trimer at a molar ratio of 1:6 (TRAP 11mer/AT trimer) and subjected to size exclusion chromatography, no peak for the TRAP-AT complex was detected (data not shown). Complex formation was, however, detected by analytical ultracentrifugation when the sample was prepared with 4-fold excess AT trimer.19 For ESI-MS, 6 times molar excess AT trimer was mixed with TRAP 11mer (wild-type) or 12mer (T3A7) to drive complex formation. In the mass spectrum of TRAP-AT complexes, shown in Figure 4, parts a and b, intense peaks of free AT 12mer were observed at m/z 3800-4400 and no peak representing free TRAP 11mer or 12mer appeared. For the wildtype TRAP spectrum, shown in Figure 4a, ions corresponding to various binding stoichiometries with AT trimer were ob(26) Shevtsov, M. B.; Chen, Y.; Gollnick, P.; Antson, A. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17600–17605.

served at m/z 5700-8000. In contrast to wild-type TRAP, T3A7 has a 12mer ring structure and showed only a single binding stoichiometry with AT, a 12mer ring TRAP and six AT trimers, as shown in Figure 4b and Table 2. Broad peaks of 30+, 29+, and 28+ ions of the complex were observed, which correspond to one TRAP 12mer with 12 Trp and 6 copies of AT trimer, with a total molecular mass of 209 880.5 ± 26.8 (theoretical mass: 208 065.3). The observed and theoretical masses of the wild-type TRAP-AT complexes are summarized in Table 2. As shown in the expanded mass spectrum (Figure 4a), peaks representing at least four complexes were observed. Peaks at m/z 6814.3, 7049.9, and 7301.1 correspond to 30+, 29+, and 28+ ions of the complex A with molecular mass of 204 406.8 ± 8.8, a complex of a TRAP 12mer ring with 12 Trp residues and 6 AT trimers (theoretical mass 204 229.0). Complex B corresponds to a TRAP 11mer ring with 11 Trp and 5 AT trimers (theoretical mass 178 638.5), complex C to TRAP 11mer with 11 Trp and 4 AT trimers (theoretical mass 161 492.5), and complex D to TRAP 11mer with 11 Trp and 3 AT trimers (theoretical mass 144 347.5). This indicates that AT trimer is the minimum unit to interact with TRAP, and only TRAP 12mer rings can provide six binding sites for AT trimers. The TRAP 11mer ring can bind a maximum of five AT trimers. Since the crystal structure of a TRAP 12mer ring and six AT trimers shows that each AT trimer interacts with two neighboring TRAP subunits, it is to be expected that the 11mer ring can bind only five AT trimers. The presence of complexes with only three or four AT trimers suggests that AT binding to the TRAP 11mer ring is not strongly cooperative. The experimental mass values of the wild-type TRAP-AT complexes are very close to those expected with Trp molecules bound, as shown in Table 2. Peaks of the TRAP-AT complexes were broadened, especially for the T3A7-AT complexes, presumably due to many attached molecules of ammonium acetate or Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Figure 4. NanoESI mass spectra of the solutions of wild-type TRAP (11mer)/AT trimer ) 1:6 in 1 M ammonium acetate (a), mutant TRAP (12mer)/AT trimer ) 1:6 in 500 mM ammonium acetate (b), and free AT in 500 mM ammonium acetate (c). Spectra were obtained at the cone voltage of 70 V and collision voltage of 20 V (a) or 30 V (b and c). An expanded mass spectrum of wild-type TRAP-AT, obtained at cone and collision voltages of 100 and 40 V, is also shown with the assignment of peaks observed in the mass range of m/z 5500-7700. A28+, A29+, and A30+ correspond to 28+, 29+, and 30+ ions of a TRAP 12mer ring with 12 Trp residues and 6 AT trimers; B25+, B26+, B27+, B28+, and B29+ correspond to 25+, 26+, 27+, 28+, and 29+ ions of a TRAP 11mer ring with 11 Trp residues and 5 AT trimers; C24+, C25+, and C26+ correspond to 24+, 25+, and 26+ ions of a TRAP 11mer ring with 11 Trp residues and 4 AT trimers; D23+, D24+, and D25+ correspond to 23+, 24+, and 25+ ions of a TRAP 11mer ring with 11 Trp residues and 3 AT trimers, respectively. The expanded mass spectrum in the upper panel is a modified form of Figure 5 in our previous article (ref 19). Adapted from ref 19. Copyright 2009 National Academy of Sciences.

Table 2. Observed and Theoretical masses of the TRAP-AT Complex obsd mass (charge number)a

exptl mass of the molecule (Da)a

theor mass with Trp (Da)

wild-type TRAP 12mer + six copies of AT 3mer

6814.3 (30+) 7049.9 (29+) 7301.1 (28+)

204 406.8 ± 8.8

204 229.0

B

wild-type TRAP 11mer + five copies of AT 3mer

6164.9 (29+) 6383.7 (28+) 6620.9 (27+) 6875.4 (26+) 7150.8 (25+)

178 737.2 ± 12.1

178 638.5

C

wild-type TRAP 11mer + four copies of AT 3mer

6214.2 (26+) 6463.6 (25+) 6733.8 (24+)

161 591.0 ± 3.1

161 492.5

D

wild-type TRAP 11mer + three copies of AT 3mer

5775.7 (25+) 6015.8 (24+) 6279.6 (23+)

144 380.0 ± 20.2

144 347.5

E

T3A7 12mer + six copies of AT 3mer

6995.7 (30+) 7239.4 (29+) 7496.1 (28+)

209 880.5 ± 26.8

208 065.3

label A

a

complex components

“obsd mass” corresponds to the most intense peak for each charge state and was used for the calculation of “exptl mass of the molecule”.

water, and the experimental mass of the complex was about 1800 Da larger than the theoretical value. DISCUSSION Binding of Tryptophan to TRAP. The MS and MS/MS experiments of McCammon et al. showed that B. subtilis TRAP 11mer and 12mer are bound with 6, 11, or 12 molecules of 2224


Trp.20 Our nanoESI mass spectra of B. stearothermophilus TRAP indicated that the most intense peak corresponds to TRAP 11mer for wild-type and TRAP 12mer for T3A7. In both cases the protein is not fully saturated with Trp, and both spectra show satellite peaks with mass difference of a few Trp molecules. The differences between the previous mass spectra and the results presented here may be due to differences in

the sample preparation, the measurement conditions of mass spectra, and differences between TRAP from B. subtilis and B. stearothermophilus, and these three possibilities are discussed in turn below. The samples for the previous study were prepared by mixing TRAP and Trp at a molar ratio of 1:4 (per monomer of TRAP) in 20 mM ammonium acetate,20 whereas the samples in the present study were prepared by extensive dialysis of the purified TRAP protein from 50 mM Tris-HCl (pH 8.5), 100 mM NaCl buffer to 50 mM ammonium acetate without addition of Trp to the solution. In the present study, the TRAP protein was expressed in E. coli cells and purified by column chromatography, as described in the Experimental Section. No additional Trp was added to the sample solution prior to dialysis for nanoESI-MS of TRAP. As discussed below, the mass spectra of TRAP-RNA or TRAP-AT complexes indicated that each TRAP subunit in the complex seems to hold a Trp, although no excess Trp was present in the solution. Another possible reason for the difference is the experimental conditions of mass measurements. There was no significant difference between the cone and collision voltages of the two studies, but the pressure in the collision cell was quite different. In our study the pressure of the quadrupole ion guide was set at 8 × 10-3 Pa, about 100-fold lower than that in the work of McCammon et al.20 Our instrument is an unmodified commercially available machine, whereas McCammon et al. employed an upgraded model capable of reaching much higher pressure. This is ideal for the analysis of large macromolecular complexes since collisional cooling at higher pressure prevents dissociation of the ligands. Dissociation of ligands from the complex might not have completely been avoided in the present study. Finally differences between our spectra and those of McCammon et al. may arise from difference between TRAP from B. subtilis and B. stearothermophilus. Trp binding to B. stearothermophilus TRAP is not cooperative, whereas binding to B. subtilis TRAP is shown to be cooperative by isothermal titration calorimetry.27 For B. subtilis wild-type TRAP, a small proportion of 12mer was seen together with the expected 11mer ring. Both TRAP 11mer and 12mer rings were bound only with 6, 11, or 12 molecules of Trp. No TRAP rings were observed to be liganded with any other number of Trp ligands in the mass spectra.20 In contrast, for B. stearothermophilus TRAP, we could observe signals representing the TRAP ring bound with 1-4 Trp molecules (Figure 1a, inset). This is consistent with other evidence that binding of Trp to the TRAP protein is cooperative in B. subtilis TRAP, whereas it is not cooperative in B. stearothermophilus TRAP. Since Trp binds to TRAP subunits with a 1:1 ratio at reasonably high affinity (Kd ) 0.34 ± 0.06 µM27), minimal losses of ligand were expected during dialysis at high protein concentration. For nanoESI-MS of TRAP-RNA or TRAP-AT complexes, sample solutions were prepared as follows: (1) TRAP solution containing 0.5 mM Trp was dialyzed against ammonium acetate buffer. (2) RNA or AT solutions were dialyzed against ammonium acetate buffer. (3) The TRAP (27) Heddle, J. G.; Okajima, T.; Scott, D. J.; Akashi, S.; Park, S. Y.; Tame, J. R. J. Mol. Biol. 2007, 371, 154–167.

solution was mixed with RNA or AT solutions without addition of Trp. Thus, the final solutions of TRAP-RNA or TRAP-AT do not contain excess free Trp, but the mass spectra of TRAP-RNA and TRAP-AT suggested that Trp residues are included in the complexes. Our mass spectra show that B. stearothermophilus TRAP can retain bound Trp in the gas phase when RNA or AT molecules are bound around the ring, as these macromolecular ligands raise the affinity of TRAP for Trp. (Since Trp greatly increases the affinity of TRAP for these molecules, Wyman’s linkage relations demand the reverse must also be true.) Binding of TRAP to AT. The structures of both wild-type TRAP and T3A7 with AT have been determined by X-ray crystallography.19 Both complexes show a 12mer TRAP ring surrounded by six copies of AT trimer, even though wild-type TRAP in solution is expected to be an 11mer ring. The ESI mass spectrum of the wild-type TRAP-AT complex demonstrated that there are at least four complexes with different binding stoichiometries: TRAP 11mer with 9, 12, or 15 AT subunits and TRAP 12mer with 18 AT subunits. Of these four complexes, peaks for the complex of TRAP 11mer with 15 AT subunits were observed with the highest intensity. The peak intensities for the complex of TRAP 12mer and 18 AT subunits were relatively low, as shown in Figure 4. Since the molecular sizes and biophysical characteristics of these complexes are close, the peak intensities can be assumed to reflect the relative abundance of each complex. This implies the TRAP11/AT15 complex is the most abundant in solution, with the TRAP12/AT18 complex observed in crystals being relatively rare. Since the buffer conditions for ESI-MS and crystallization are not identical, it is possible that the different binding stoichiometries for the TRAP-AT complex indicated by these two methods are a reflection of the pH and ionic strength. The TRAP 11mer is, however, extremely stable, and there is no evidence to suggest the oligomeric state changes with pH or ionic strength alone. The crystal structures of the TRAP-AT complex at pH 7.0 and 9.0 are essentially identical. Surface plasmon resonance experiments show that at neutral pH AT binds to 11mer and 12mer TRAP with similar affinity (unpublished results). Our present results therefore strongly suggest that crystallography provides a highly selective view of the mixture, with only one highly symmetrical component forming crystals due to its ready packing into a three-dimensional lattice, whereas ESI-MS can identify all the complexes in solution and provide an approximate estimate of relative abundance. In the absence of AT, wild-type B. stearothermophilus TRAP shows very little 12mer ring formation, but the crystal structure of wild-type TRAP with AT showed 12mer ring TRAP complexed with six AT trimers. This implies a remodeling of the TRAP ring due to the relatively weak TRAP-AT interaction and crystal lattice packing. 11mer and 12mer ring forms are in equilibrium in solution, and addition of AT pushes the equilibrium toward the 12mer ring form which has a greater binding capacity for AT. The TRAP 12mer-AT 18mer complex is selectively pulled out from the solution by crystallization. The crystallographic structures of the TRAP-AT complex could be interpreted alone to indicate, incorrectly, that TRAP remodeling from an 11mer to a 12mer is required for AT binding. Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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This hypothesis is disproved by measuring mass averages with analytical centrifugation. Accurate mass measurement of the TRAP-AT complexes further reveals the presence of a number of complexes in solution and gives an unambiguous picture of AT binding as trimers to TRAP rings of 11 or 12 subunits. CONCLUSION For the characterization of macromolecular complexes, ESIMS data have proved essential in analyzing analytical ultracentrifugation studies of the same system, in which several different complexes have similar molecular weight and sedimentation

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characteristics. The exquisite precision of ESI-MS can reveal similar complexes present in solution, which cannot always be completely accomplished by X-ray crystallography alone. ACKNOWLEDGMENT This work was supported by Grants-in-Aid for Scientific Research (MEXT). Received for review November 6, 2008. Accepted January 13, 2009. AC802354J

RNA and Protein Complexes of trp RNA-Binding Attenuation Protein

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