Dynamics of Reassembled Thioredoxin Studied by Magic Angle

Sep 8, 2009 - Solid-state NMR spectroscopy can be used to probe internal protein dynamics in the absence of the overall molecular tumbling. In this st...
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Dynamics of Reassembled Thioredoxin Studied by Magic Angle Spinning NMR: Snapshots from Different Time Scales Jun Yang,†,§ Maria Luisa Tasayco,‡ and Tatyana Polenova*,† Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716, and Department of Chemistry, The City College of New York, ConVent AVenue at 138th Street, New York, New York 10031 Received May 9, 2009; E-mail: [email protected]

Abstract: Solid-state NMR spectroscopy can be used to probe internal protein dynamics in the absence of the overall molecular tumbling. In this study, we report 15N backbone dynamics in differentially enriched 1-73(U-13C,15N)/74-108(U-15N) reassembled thioredoxin on multiple time scales using a series of 2D and 3D MAS NMR experiments probing the backbone amide 15N longitudinal relaxation, 1H-15N dipolar order parameters, 15N chemical shift anisotropy (CSA), and signal intensities in the temperature-dependent and 1 H T2′-filtered NCA experiments. The spin-lattice relaxation rates R1 (R1 ) 1/T1) were observed in the range from 0.012 to 0.64 s-1, indicating large site-to-site variations in dynamics on pico- to nanosecond time scales. The 1H-15N dipolar order parameters, 〈S〉, and 15N CSA anisotropies, δσ, reveal the backbone mobilities in reassembled thioredoxin, as reflected in the average 〈S〉 ) 0.89 ( 0.06 and δσ ) 92.3 ( 5.2 ppm, respectively. From the aggregate of experimental data from different dynamics methods, some degree of correlation between the motions on the different time scales has been suggested. Analysis of the dynamics parameters derived from these solid-state NMR experiments indicates higher mobilities for the residues constituting irregular secondary structure elements than for those located in the R-helices and β-sheets, with no apparent systematic differences in dynamics between the R-helical and β-sheet residues. Remarkably, the dipolar order parameters derived from the solid-state NMR measurements and the corresponding solution NMR generalized order parameters display similar qualitative trends as a function of the residue number. The comparison of the solid-state dynamics parameters to the crystallographic B-factors has identified the contribution of static disorder to the B-factors. The combination of longitudinal relaxation, dipolar order parameter, and CSA line shape analyses employed in this study provides snapshots of dynamics and a new insight on the correlation of these motions on multiple time scales.

Introduction

Protein dynamics is essential for various biological functions, such as enzyme catalysis,1-4 ion transport across biological membranes by ion channel proteins,5-9 signaling,10,11 and †

University of Delaware. The City College of New York. Current address: Institut fu¨r Biophysicalische Chemie, Goethe Universita¨t, Max-von-Laue-Str. 9, Biozentrum N202, 60438 Frankfurt am Main, Germany. (1) McDermott, A.; Polenova, T. Curr. Opin. Struct. Biol. 2007, 17, 617– 622. (2) Butterwick, J. A.; Palmer, A. G. Protein Sci. 2006, 15, 2697–2707. (3) Loria, J. P.; Berlow, R. B.; Watt, E. D. Acc. Chem. Res. 2008, 41, 214–221. (4) Kern, D. Biochemistry 2003, 42, 8605–8605. (5) Baker, K. A.; Tzitzilonis, C.; Kwiatkowski, W.; Choe, S.; Riek, R. Nat. Struct. Mol. Biol. 2007, 14, 1089–1095. (6) Varga, K.; Tian, L.; McDermott, A. E. Biochim. Biophys. Acta, Proteins Proteomics 2007, 1774, 1604–1613. (7) Lange, A.; Giller, K.; Hornig, S.; Martin-Eauclaire, M. F.; Pongs, O.; Becker, S.; Baldus, M. Nature 2006, 440, 959–962. (8) Ader, C.; Schneider, R.; Hornig, S.; Velisetty, P.; Wilson, E. M.; Lange, A.; Giller, K.; Ohmert, I.; Martin-Eauclaire, M. F.; Trauner, D.; Becker, S.; Pongs, O.; Baldus, M. Nat. Struct. Mol. Biol. 2008, 15, 605–612. (9) Roux, B.; Allen, T.; Berneche, S.; Im, W. Q. ReV. Biophys. 2004, 37, 15–103. ‡ §

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formation of misfolded proteins.12-14 Internal motions in proteins range from small amplitude pico- to nanosecond scale movements to large conformational changes occurring on the time scales of microseconds to seconds. Characterization of dynamic behavior in proteins and protein assemblies on a wide range of time scales is necessary not only because insight may be gained into the functional significance of motions in a particular system but also because this knowledge may help establish general principles underlying dynamic properties of proteins as a function of the overall architecture and environment. NMR is a powerful approach to study protein dynamics on multiple time scales from picoseconds to seconds.15-27 Solidstate NMR methods can probe internal dynamics in the absence of the overall molecular tumbling, using anisotropic line shape analysis and relaxation measurements.28-31 In solid-state NMR, (10) Tsao, D. H. H.; Hum, W. T.; Hsu, S.; Malakian, K.; Lin, L. L. J. Biomol. NMR 2007, 39, 337–342. (11) Abdulaev, N. G.; Ramon, E.; Ngo, T.; Brabazon, D. M.; Marino, J. P.; Ridge, K. D. FASEB J. 2007, 21, A982–A983. (12) Sprangers, R.; Kay, L. E. Nature 2007, 445, 618–622. (13) Barducci, A.; Chelli, R.; Procacci, P.; Schettino, V.; Gervasio, F. L.; Parrinello, M. J. Am. Chem. Soc. 2006, 128, 2705–2710. (14) Bollen, Y. J. M.; Kamphuis, M. B.; van Mierlo, C. P. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4095–4100. (15) Cole, H. B. R.; Torchia, D. A. Chem. Phys. 1991, 158, 271–281. (16) Torchia, D. A. Annu. ReV. Biophys. Bioeng. 1984, 13, 125–144. 10.1021/ja9037802 CCC: $40.75  2009 American Chemical Society

Thioredoxin Dynamics from MAS NMR Spectroscopy

the anisotropic resonance frequency depends on the orientation of the molecular frame with respect to the static magnetic field. The motions with the correlation times faster than the inverse of the corresponding anisotropic interactions partially average the orientational dependence and thus decrease the width of the anisotropic spectra. These motionally narrowed spectra contain information about the amplitude and the correlation time of the motions. For example, the anisotropic 2H line shapes determined by quadrupolar, dipolar, and chemical shift anisotropy (CSA) interactions were generally used to probe dynamics in peptides and proteins by solid-state NMR.29,30,32-34 Static deuterium NMR measurements have been widely used to study motions in proteins, but it is difficult with this approach to investigate multiple sites simultaneously to extract residue-specific dynamics because of signal overlap.33 Heteronuclear (13C,15N) dipolar coupling and CSA line shape analysis can also be used to detect internal dynamics in proteins by solid-state NMR. For an axially symmetric dipolar interaction, the ratio of the motionally narrowed dipolar coupling to the rigid-limit coupling is defined as the order parameter. In the ideal rigid environment in the absence of molecular dynamics, the order parameter is 1, while 0 represents the fully motionally averaged isotropic orientation. The dipolar order parameter is thus a measure of the amplitude of the rotation of the internuclear bond vector. In the case of CSA, the correlation time giving rise to the fast limit motions that would reduce the anisotropic line width depends on the nucleus type and on the static magnetic field. In the past decade, the advances in the preparation of uniformly and differentially enriched protein samples for solidstate NMR spectroscopy35-42 and the development of the dipolar (17) Nicholson, L. K.; Teng, Q.; Cross, T. A. J. Mol. Biol. 1991, 218, 621–637. (18) Bogusky, M. J.; Schiksnis, R. A.; Leo, G. C.; Opella, S. J. J. Magn. Reson. 1987, 72, 186–190. (19) Gall, C. M.; Cross, T. A.; Diverdi, J. A.; Opella, S. J. Proc. Nat. Acad. Sci. U.S.A. 1982, 79, 101–105. (20) Williams, J. C.; McDermott, A. E. Biochemistry 1995, 34, 8309– 8319. (21) Palmer, A. G.; Williams, J.; McDermott, A. J. Phys. Chem. 1996, 100, 13293–13310. (22) Ishima, R.; Torchia, D. A. Nat. Struct. Biol. 2000, 7, 740–743. (23) Stone, M. J. Acc. Chem. Res. 2001, 34, 379–388. (24) Palmer, A. G. Chem. ReV. 2004, 104, 3623–3640. (25) Mittermaier, A.; Kay, L. E. Science 2006, 312, 224–228. (26) Jarymowycz, V. A.; Stone, M. J. Chem. ReV. 2006, 106, 1624–1671. (27) Kay, L. E. J. Magn. Reson. 2005, 173, 193–207. (28) Hiyama, Y.; Niu, C. H.; Silverton, J. V.; Bavoso, A.; Torchia, D. A. J. Am. Chem. Soc. 1988, 110, 2378–2383. (29) Jelinski, L. W.; Sullivan, C. E.; Torchia, D. A. Nature 1980, 284, 531–534. (30) Torchia, D. A. Methods Enzymol. 1982, 82, 174–186. (31) Tuzi, S.; Shinzawaitoh, K.; Erata, T.; Naito, A.; Yoshikawa, S.; Saito, H. Eur. J. Biochem. 1992, 208, 713–720. (32) Copie, V.; McDermott, A. E.; Beshah, K.; Williams, J. C.; Spijkerassink, M.; Gebhard, R.; Lugtenburg, J.; Herzfeld, J.; Griffin, R. G. Biochemistry 1994, 33, 3280–3286. (33) Huster, D. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 46, 79–107. (34) Krushelnitsky, A.; Reichert, D. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 47, 1–25. (35) McDermott, A.; Polenova, T.; Bockmann, A.; Zilm, K. W.; Paulsen, E. K.; Martin, R. W.; Montelione, G. T. J. Biomol. NMR 2000, 16, 209–219. (36) Bockmann, A.; Lange, A.; Galinier, A.; Luca, S.; Giraud, N.; Juy, M.; Heise, H.; Montserret, R.; Penin, F.; Baldus, M. J. Biomol. NMR 2003, 27, 323–339. (37) Marulanda, D.; Tasayco, M. L.; Cataldi, M.; Arriaran, V.; Polenova, T. J. Phys. Chem. B 2005, 109, 18135–18145. (38) Marulanda, D.; Tasayco, M. L.; McDermott, A.; Cataldi, M.; Arriaran, V.; Polenova, T. J. Am. Chem. Soc. 2004, 126, 16608–16620. (39) Yang, J.; Paramasivan, S.; Marulanda, D.; Cataidi, M.; Tasayco, M. L.; Polenova, T. Magn. Reson. Chem. 2007, 45, S73–S83.

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or/and J-based multidimensional magic angle spinning (MAS) NMR pulse sequences43-50 have led to complete resonance assignments36-40,42,51 and to determination of the full 3D structures52-57 of several globular proteins. In the meantime, the development of the dipolar recoupling techniques permitting recovery of the anisotropic interactions removed by MAS58-62 opened the field to site-specific investigations of protein dynamics.40,62-72 Combining recoupling techniques with mul(40) Franks, W. T.; Zhou, D. H.; Wylie, B. J.; Money, B. G.; Graesser, D. T.; Frericks, H. L.; Sahota, G.; Rienstra, C. M. J. Am. Chem. Soc. 2005, 127, 12291–12305. (41) Siemer, A. B.; Ritter, C.; Ernst, M.; Riek, R.; Meier, B. H. Angew. Chem., Int. Ed. 2005, 44, 2441–2444. (42) Igumenova, T. I.; McDermott, A. E.; Zilm, K. W.; Martin, R. W.; Paulson, E. K.; Wand, A. J. J. Am. Chem. Soc. 2004, 126, 6720– 6727. (43) Verel, R.; Ernst, M.; Meier, B. H. J. Magn. Reson. 2001, 150, 81– 99. (44) Hohwy, M.; Rienstra, C. M.; Griffin, R. G. J. Chem. Phys. 2002, 117, 4973–4987. (45) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. J. Chem. Phys. 1995, 103, 6951–6958. (46) Chen, L. L.; Olsen, R. A.; Elliott, D. W.; Boettcher, J. M.; Zhou, D. H. H.; Rienstra, C. M.; Mueller, L. J. J. Am. Chem. Soc. 2006, 128, 9992–9993. (47) Linser, R.; Fink, U.; Reif, B. J. Magn. Reson. 2008, 193, 89–93. (48) Lesage, A.; Auger, C.; Caldarelli, S.; Emsley, L. J. Am. Chem. Soc. 1997, 119, 7867–7868. (49) Takegoshi, K.; Nakamura, S.; Terao, T. Chem. Phys. Lett. 2001, 344, 631–637. (50) Chen, L.; Kaiser, J. M.; Polenova, T.; Yang, J.; Rienstra, C. M.; Mueller, L. J J. Am. Chem. Soc. 2007, 129, 10650-+. (51) Pauli, J.; Baldus, M.; van Rossum, B.; de Groot, H.; Oschkinat, H. ChemBioChem 2001, 2, 272–281. (52) Castellani, F.; van Rossum, B.; Diehl, A.; Schubert, M.; Rehbein, K.; Oschkinat, H. Nature 2002, 420, 98–102. (53) Zech, S. G.; Wand, A. J.; McDermott, A. E. J. Am. Chem. Soc. 2005, 127, 8618–8626. (54) Lange, A.; Becker, S.; Seidel, K.; Giller, K.; Pongs, O.; Baldus, M. Angew. Chem., Int. Ed. 2005, 44, 2089–2092. (55) Franks, W. T.; Wylie, B. J.; Schmidt, H. L. F.; Nieuwkoop, A. J.; Mayrhofer, R. M.; Shah, G. J.; Graesser, D. T.; Rienstra, C. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4621–4626. (56) Loquet, A.; Bardiaux, B.; Gardiennet, C.; Blanchet, C.; Baldus, M.; Nilges, M.; Malliavin, T.; Boeckmann, A. J. Am. Chem. Soc. 2008, 130, 3579–3589. (57) Manolikas, T.; Herrmann, T.; Meier, B. H. J. Am. Chem. Soc. 2008, 130, 3959–3966. (58) van Rossum, B. J.; de Groot, C. P.; Ladizhansky, V.; Vega, S.; de Groot, H. J. M. J. Am. Chem. Soc. 2000, 122, 3465–3472. (59) Hong, M.; Yao, X. L.; Jakes, K.; Huster, D. J. Phys. Chem. B 2002, 106, 7355–7364. (60) Chan, J. C. C.; Tycko, R. J. Chem. Phys. 2003, 118, 8378–8389. (61) Zhao, X.; Eden, M.; Levitt, M. H. Chem. Phys. Lett. 2001, 342, 353– 361. (62) Hohwy, M.; Jaroniec, C. P.; Reif, B.; Rienstra, C. M.; Griffin, R. G. J. Am. Chem. Soc. 2000, 122, 3218–3219. (63) Huster, D.; Xiao, L. S.; Hong, M. Biochemistry 2001, 40, 7662– 7674. (64) Lorieau, J. L.; McDermott, A. E. J. Am. Chem. Soc. 2006, 128, 11505–11512. (65) Lorieau, J. L.; Day, L. A.; McDermott, A. E Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 10366–10371. (66) Chevelkov, V.; Diehl, A.; Reif, B J. Chem. Phys. 2008, 128. (67) Chevelkov, V.; Zhuravleva, A. V.; Xue, Y.; Reif, B.; Skrynnikov, N. R. J. Am. Chem. Soc. 2007, 129, 12594–12595. (68) Reif, B.; Xue, Y.; Agarwal, V.; Pavlova, M. S.; Hologne, M.; Diehl, A.; Ryabov, Y. E.; Skrynnikov, N. R. J. Am. Chem. Soc. 2006, 128, 12354–12355. (69) Giraud, N.; Bockmann, A.; Lesage, A.; Penin, F.; Blackledge, M.; Emsley, L. J. Am. Chem. Soc. 2004, 126, 11422–11423. (70) Giraud, N.; Blackledge, M.; Goldman, M.; Bockmann, A.; Lesage, A.; Penin, F.; Emsley, L. J. Am. Chem. Soc. 2005, 127, 18190–18201. (71) Kandasamy, S. K.; Lee, D. K.; Nanga, R. P. R.; Xu, J.; Santos, J. S.; Larson, R. G.; Ramamoorthy, A. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 686–695. (72) Xu, J. D.; Durr, U. H. N.; Im, S. C.; Gan, Z. H.; Waskell, L.; Ramamoorthy, A. Angew. Chem., Int. Ed. 2008, 47, 7864–7867. J. AM. CHEM. SOC.

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Yang et al.

tidimensional MAS sequences for resonance assignments permits site-specific dynamics studies in uniformly and multiply isotopically labeled proteins, via relaxation and anisotropic line shape analysis.40,64-67,69,70 To date, only a few examples have been reported where protein dynamics has been investigated site-specifically by solidstate NMR spectroscopy. Gieaud et al. measured backbone amide 15N spin-lattice relaxation times of Crh using U-13C,15N labeled microcrystalline protein sample and proposed a theoretical model for the quantitative analysis of the MAS NMR longitudinal relaxation time.69,70 Chevelkov et al. measured 15N T1 of microcrystalline SH3 domain of R-spectrin at several magnetic fields using perdeuterated samples.66 They provided quantitative analysis based on the combination of solid-state and solution NMR T1 relaxation data and concluded that the relaxation times determined by both methods correlate strongly.67,68 Huster et al., Lorieau et al., and Franks et al. studied sitespecifically the local dynamics of several proteins using 1H-13C and 1H-15N dipolar order parameters.40,63-65,73,74 In addition, 3D 15N CSA measurements conducted by Wylie et al. showed that backbone dynamics can be probed by recording the 15N CSA tensors.75 The spin-lattice relaxation time measurements reflect the rate of motion on the time scales of pico- to nanoseconds, whereas 1H-13C and 1H-15N dipolar order parameters and 15N CSA tensors are sensitive to the motional amplitude in the time window of sub-microsecond. To date, there have been no reports of site-specific protein dynamics studied by a combination of relaxation-rate and motionalamplitude measurements performed on the same protein and under the same conditions by solid-state NMR. In this work, we report backbone amide 15N dynamics in the 1-73 N-terminal fragment of the 1-73(U-13C,15N)/74-108(U15 N) reassembled thioredoxin investigated by combined 2D and 3D MAS NMR measurements of 15N spin-lattice relaxation rates, 1H-15N dipolar order parameters, 15N CSA, as well as the signal intensities in the variable-temperature and amide 1HT2′-filtered NCA 2D experiments. Thioredoxin is a 11.7 kDa protein, whose tertiary structure, called a “thioredoxin fold”, is composed of five β-sheet and four R-helices. The 3D structures of thioredoxin are available from the X-ray crystallography76 and solution NMR.77 The dynamics of the backbone amide 15N atoms of thioredoxin has been extensively studied by solution NMR.78 In our previous investigations, we obtained resonance assignments of intact and reassembled thioredoxin samples using MAS NMR spectroscopy37-39 and developed NMR experiments for studies of interfaces of differentially enriched protein assemblies, using thioredoxin as an example.79 In this report, we present backbone dynamics of thioredoxin on multiple time scales. Analysis of the dynamics parameters based on the measurements of spin-lattice relaxation rates, dipolar order parameters, and CSA line shapes reveals surprising correlations (73) Barre, P.; Yamaguchi, S.; Saito, H.; Huster, D. Eur. Biophys. J. Biophys. Lett. 2003, 32, 578–584. (74) Vogel, A.; Katzka, C. P.; Waldmann, H.; Arnold, K.; Brown, M. F.; Huster, D. J. Am. Chem. Soc. 2005, 127, 12263–12272. (75) Wylie, B. J.; Franks, W. T.; Rienstra, C. M. J. Phys. Chem. B 2006, 110, 10926–10936. (76) Katti, S. K.; Lemaster, D. M.; Eklund, H. J. Mol. Biol. 1990, 212, 167–184. (77) Dyson, H. J.; Gippert, G. P.; Case, D. A.; Holmgren, A.; Wright, P. E. Biochemistry 1990, 29, 4129–4136. (78) Stone, M. J.; Chandrasekhar, K.; Holmgren, A.; Wright, P. E.; Dyson, H. J. Biochemistry 1993, 32, 426–435. (79) Yang, J.; Tasayco, M. L.; Polenova, T. J. Am. Chem. Soc. 2008, 130, 5798–5807. 13692

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between motions occurring on different time scales, as well as correlations between dynamic behavior and the tertiary structure of the protein. In addition, dynamics parameters from MAS NMR exhibit interesting correlations with the crystallographic B-factors. Finally, dipolar order parameters from MAS NMR measurements and order parameters derived from the Lipari-Szabo analysis of solution NMR relaxation data indicate similar qualitative trends as a function of the residue number. This study demonstrates that the combined measurement of relaxation and anisotropic line shape parameters provides snapshots of thioredoxin dynamics across a wide range of time scales and yields new insights on the correlations between motions occurring on these different time scales. This solid-state NMR approach yields a comprehensive view of dynamics in reassembled thioredoxin and is broadly applicable to other proteins and protein assemblies. Experimental Section Sample Preparation. Overexpression and purification protocols for U-13C,15N isotopically enriched thioredoxin were described in previous reports.38,80 Preparation of differentially enriched 1-73(U13 15 C, N)/74-108(U-15N) thioredoxin reassembly was reported in detail previously.39 In brief, a proteolytic cleavage site was engineered at Arg-73. 13C,15N- and 15N-enriched purified thioredoxin was prepared, and each sample was cleaved into two complementary fragments: one fragment containing the N-terminal residues 1-73 and the other containing the C-terminal residues 74-108. The fragments were purified, and the 1-73(U-13C,15N) fragment was reconstituted with the 74-108(U-15N) fragment. For solid-state NMR experiments, a sample of reassembled 1-73(U-13C,15N)/ 74-108(U-15N) thioredoxin was prepared by controlled precipitation of 70 mg/mL reassembled thioredoxin in 10 mM phosphate buffer (pH 7.0) from PEG-4000, which is accomplished by the slow addition of a PEG-4000 solution containing 10 mM NaCH3COO and 1 mM NaN3 (pH 3.5), as described previously.38 The sample was centrifuged to remove the supernatant. 11 mg of hydrated thioredoxin precipitate were packed into a 3.2 mm Varian rotor and sealed using an upper spacer and a top spinner. Solid-State NMR Spectroscopy. All NMR spectra were acquired on a 14.1 T narrow bore Varian InfinityPlus spectrometer equipped with a 3.2 mm triple-resonance T3 MAS probe. The MAS frequency in all experiments was 10 kHz controlled by a Varian MAS controller to within (0.002 kHz. A PbNO3 temperature sensor was used as a temperature calibration compound for this probe at different MAS frequencies.81 The actual temperature at the sample was maintained at 288 ( 0.5 K (15 °C) throughout the experiments using the Varian temperature controller, except in the variabletemperature experiments, where additional spectra were acquired at 263 K (-10 °C) and 238 K (-35 °C). 1H, 13C, and 15N chemical shifts were referenced with respect to DSS, with adamantane and ammonium chloride used as external referencing standards.82 For most of the experiments, the pulse lengths were 2.9 µs (1H), 5 µs (13C), and 5 µs (15N). The 1H-X (X ) 15N or 13C) cross-polarization (CP) employed a tangent amplitude ramp (80-100%), the 1H radio frequency field was 50 kHz, and the center of the ramp on the heteronucleus was Hartmann-Hahn matched to the first spinning sideband. The band-selective magnetization transfer from 15N to 13 R C was realized using 5.2 ms SPECIFIC-CP83 with tangent amplitude ramp and 27, 17, and 100 kHz RF power in 15N, 13C and 1H, respectively. The 1H decoupling power of ∼80 kHz was (80) Yu, W.; Gibaja, V.; Arevalo, E.; Petit, M. C.; Li, J. H.; Tasayco, M. L. Biophys. J. 1997, 72, Wp386–Wp386. (81) Neue, G.; Dybowski, C. Solid State Nucl. Magn. Reson. 1997, 7, 333–336. (82) Morcombe, C. R.; Zilm, K. W. J. Magn. Reson. 2003, 162, 479– 486. (83) Baldus, M.; Petkova, A. T.; Herzfeld, J.; Griffin, R. G. Mol. Phys. 1998, 95, 1197–1207.

Thioredoxin Dynamics from MAS NMR Spectroscopy

typically used during the acquisition and evolution periods in the 2D and 3D experiments. For the 15N spin-lattice relaxation time (T1) measurement, a basic 2D NCA experiment was modified with the 90-τ-90 element inserted following the 1H/15N CP and before the 15N t1 evolution period.69 The relaxation curves were acquired using the delays of 0.1, 1, 3, 7, and 15 s. To compensate for the minor instability of the amplifiers and for the slight detuning of the probe during the 2D measurements, we acquired 2D NCA spectra with a 0.1 s longitudinal delay before and after every measurement and renormalized the intensity of the resolved peaks according to the average intensity of the peaks in these control spectra, following previous reports.69,70 In the 3D DIPSHIFT experiments, the DIPSHIFT dipolar recoupling period84 was incorporated into the basic 2D NCA sequence; this combination permits to resolve cross peaks for individual residues. In DIPSHIFT, an RN-type recoupling block, R1817,61 was employed for the recoupling of the 1H-15N dipolar coupling and at the same time for the suppression of the 1H-1H homonuclear dipolar interactions. For the recoupling of the 15N CSA, a 3D ROCSA experiment was conducted.60,75 For the ROCSA period, a C221 POST block was used with (a, b) ) (0.0329, 0.467) and 1 rotor period (100 µs) increment per t1 point. During ROCSA, a 10 µs 13C π pulse with XY-8 phase cycling scheme85 was introduced in the middle of every rotor period on the 13C channel, and 110 kHz CW decoupling was employed on the 1H channel. Haeberlen-Mehring-Spiess convention is followed in the definition of the CSA tensor. δiso is the isotropic chemical shift defined as δiso ) 1/3(δxx + δyy + δzz). δxx, δyy, and δzz are the principal components of the CSA tensor defined as |δzz - δiso| g |δxx - δiso| g |δyy - δiso|. δσ is the reduced anisotropy of the CSA tensor determining the breadth of the tensor (throughout the text, we will refer to δσ as anisotropy) defined as δσ ) δzz δiso, and η is the asymmetry parameter of the CSA tensor determining the deviation from the axial symmetry, defined as ησ ) (δyy - δxx)/(δzz - δiso). The chemical shift assignments of 1-73(U-13C,15N)/74-108(U15 N) reassembled thireodoxin have been reported by us previously.39 Nearly complete backbone and side chain assignments have been made except for residues at the N-terminus including S1, D2, and K3. Data Processing and Analysis. All spectra were processed in NMRpipe86 and analyzed in Sparky.87 90° or 60° shifted sine bell followed by a Lorentzian-to-Gaussian transformation were applied in both dimensions; forward linear prediction to twice the number of the original data points was employed in the indirect dimension followed by zero filling to twice the total number of points prior to Fourier transformation and phasing. The chemical shift values and cross peak intensities in the 2D NCA spectra were obtained automatically in Sparky. The 15N-1H dipolar and 15N CSA line shapes in 3D spectra were extracted from the 2D NCA planes using the automatic peak picking procedure “autofit” in nmrPipe. The spin-lattice relaxation curves corresponding to the individual residues were obtained from five 2D NCA experiments with varying spin-lattice relaxation delays (see above) and subsequently fitted to the following equation I ) I0 exp(-R1t), assuming monoexponential decays (as justified by the experimental data). The uncertainty in the R1 values was estimated from the standard errors of the fitting. The H-N dipolar coupling constants were extracted by the numerical simulations of the dipolar line shapes using SPINEVO(84) Munowitz, M.; Aue, W. P.; Griffin, R. G. J. Chem. Phys. 1982, 77, 1686–1689. (85) Holl, S. M.; Mckay, R. A.; Gullion, T.; Schaefer, J. J. Magn. Reson. 1990, 89, 620–626. (86) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277–293. (87) Goddard, T. D.; Kneller, D. G. SPARKY 3; University of California, San Francisco: San Francisco, CA.

ARTICLES

LUTION.88 The 15N CSA line shapes were simulated in SPINEVOLUTION using two-parameter fits, with chemical shift anisotropy, δσ, and asymmetry parameter, η, being adjustable parameters. The first guess values were derived from the visual inspection of the lineshapes. Subsequently, a 20 × 20 grid was constructed from 20 δσ values using an increment of 1 ppm and 20 η values using an increment of 0.01, and calculations were performed to yield the minimal χ2. A contour plot of χ2 as a function of δσ and η was built to get the best-fit values of δσ with minimum χ2. The uncertainties associated with δσ and η were estimated from the corresponding values of δσ and η giving χ2 ) 2χmin2.89 Results Temperature Dependence of Heteronuclear NCA Correlation Spectra. A broad-range temperature screen is

typically performed for identification of optimal resolution and sensitivity of the MAS NMR spectra in microcrystalline proteins. When solvent translational diffusion or fluctuating motions occur at rates comparable to the frequencies of MAS and/or 1H decoupling, which are typically in the range of 10-100 kHz, these motions interfere with MAS and/or proton decoupling, leading to broadening of the NMR signals and significant intensity loss. In addition, large-amplitude motions with correlation times shorter than the inverse of the X-H dipolar coupling cause a decrease of the T1F of the 1H and X spins and a reduction of the H-X cross-polarization efficiency. The broadening of the signals and the suppression of the intensity are therefore a qualitative manifestation of the presence of motions occurring on the time scales of 10-100 µs and