Abstract
Protein–protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble, and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility, or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR-based methods to study several protein complexes. In this chapter, we discuss the general SSNMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies, and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection, and data analysis.
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References
Yool A. J. (2007) Aquaporins: Multiple roles in the central nervous system. Neuroscientist 13, 470–485.
Vale R. D. (2003) The molecular motor toolbox for intracellular transport. Cell 112, 467–480.
Grunewald K. & Cyrklaff M. (2006) Structure of complex viruses and virus-infected cells by electron cryo tomography. Curr. Opin. Microbiol. 9, 437–442.
Klein K. C., Reed J. C., & Lingappa J. R. (2007) Intracellular destinies: Degradation, targeting, assembly, and endocytosis of HIV gag. AIDS Rev. 9, 150–161.
Uysal H., et al. (2010) Antibodies to citrullinated proteins: molecular interactions and arthritogenicity. Immunol. Rev. 233, 9–33.
Goldbourt A., Gross B. J., Day L. A., & McDermott A. E. (2007) Filamentous phage studied by magic-angle spinning NMR: Resonance assignment and secondary structure of the coat protein in Pf1. J. Am. Chem. Soc. 129, 2338–2344.
Hong M. (2007) Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR Spectroscopy. J. Phys. Chem. B 111, 10340–10351.
Lange A., et al. (2006) Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962.
Porcelli F., Buck-Koehntop B. A., Thennarasu S., Ramamoorthy A., & Veglia G. (2006) Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers, determined by NMR spectroscopy. Biochemistry 45, 5793–5799.
Zheng Z., Yang R., Bodner M. L., & Weliky D. P. (2006) Conformational flexibility and strand arrangements of the membrane-associated HIV fusion peptide trimer probed by solid-state NMR spectroscopy. Biochemistry 45, 12960–12975.
Lewandowski J. R., De Paepe G., & Griffin R. G. (2007) Proton assisted insensitive nuclei cross polarization. J. Am. Chem. Soc. 129, 728–729.
Chimon S. & Ishii Y. (2005) Capturing intermediate structures of Alzheimer’s beta-amyloid, A beta(1–40), by solid-state NMR spectroscopy. J. Am. Chem. Soc. 127, 13472–13473.
Jaroniec C. P., et al. (2004) High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy. Proc. Natl. Acad. Sci. USA 101, 711–716.
Petkova A. T., et al. (2004) Solid state NMR reveals a pH-dependent antiparallel beta-sheet registry in fibrils formed by a beta-amyloid peptide. J. Mol. Biol. 335, 247–260.
Shewmaker F., Wickner R. B., & Tycko R. (2006) Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure. Proc. Natl. Acad. Sci. USA 103, 19754–19759.
Siemer A. B., et al. (2006) Observation of highly flexible residues in amyloid fibrils of the HET-s prion. J. Am. Chem. Soc. 128, 13224–13228.
Tycko R. (2006) Molecular structure of amyloid fibrils: insights from solid-state NMR. Q. Rev. Biophys. 39, 1–55.
Han Y., et al. (2010) Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies. J. Am. Chem. Soc. 132, 1976–1987.
Sun S. J., Siglin A., Williams J. C., & Polenova T. (2009) Solid-State and Solution NMR Studies of the CAP-Gly Domain of Mammalian Dynactin and Its Interaction with Microtubules. J. Am. Chem. Soc. 131, 10113–10126.
Etzkorn M., Bockmann A., Lange A., & Baldus M. (2004) Probing molecular interfaces using 2D magic-angle-spinning NMR on protein mixtures with different uniform labeling. J. Am. Chem. Soc. 126, 14746–14751.
Marulanda D., et al. (2004) Magic angle spinning solid-state NMR spectroscopy for structural studies of protein interfaces. Resonance assignments of differentially enriched Escherichia coli thioredoxin reassembled by fragment complementation. J. Am. Chem. Soc. 126, 16608–16620.
Yang J., et al. (2007) Magic angle spinning NMR spectroscopy of thioredoxin reassemblies. Magn. Reson. Chem. 45, S73-S83.
Castellani F., et al. (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420, 98–102.
Hong M. & Jakes K. (1999) Selective and extensive 13 C labeling of a membrane protein for solid-state NMR investigations. J. Biomol. NMR 14, 71–74.
Muchmore D. C., McIntosh L. P., Russell C. B., Anderson D. E., & Dahlquist F. W. (1989) Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 177, 44–73.
Marulanda D., Tasayco M. L., Cataldi M., Arriaran V., & Polenova T. (2005) Resonance assignments and secondary structure analysis of E. coli thioredoxin by magic angle spinning solid-state NMR spectroscopy. J. Phys. Chem. B 109, 18135–18145.
Yang J., Tasayco M. L., & Polenova T. (2008) Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies. J. Am. Chem. Soc. 130, 5798–5807.
Yang J., Tasayco M. L., & Polenova T. (2009) Dynamics of Reassembled Thioredoxin Studied by Magic Angle Spinning NMR: Snapshots from Different Time Scales. J. Am. Chem. Soc. 131, 13690–13702.
Franks W., Kloepper K., Wylie B., & Rienstra C. (2007) Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins. J. Biomol. NMR 39, 107–131.
Andrew E. R., Bradbury A., & Eades R. G. (1958) Nuclear Magnetic Resonance Spectra from a Crystal Rotated at High Speed. Nature 182, 1659–1659.
Schaefer J., McKay R. A., & Stejskal E. O. (1979) Double-cross-polarization NMR of solids. J. Magn. Reson. 34, 443–447.
Baldus M., Petkova A. T., Herzfeld J., & Griffin R. G. (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol. Phys. 95, 1197–1207.
Szeverenyi N. M., Sullivan M. J., & Maciel G. E. (1982) Observation of spin exchange by two-dimensional fourier transform 13C cross polarization-magic-angle spinning. J. Magn. Reson. 47, 462–475.
Takegoshi K., Nakamura S., & Terao T. (2001) 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631–637.
Verel R., Baldus M., Ernst M., & Meier B. H. (1998) A homonuclear spin-pair filter for solid-state NMR based on adiabatic-passage techniques. Chem. Phys. Lett. 287, 421–428.
Bennett A. E., et al. (1998) Homonuclear radio frequency-driven recoupling in rotating solids. J. Chem. Phys. 108, 9463–9479.
Hohwy M., Rienstra C. M., Jaroniec C. P., & Griffin R. G. (1999) Fivefold symmetric homonuclear dipolar recoupling in rotating solids: Application to double quantum spectroscopy. J. Chem. Phys. 110, 7983–7992.
Ernst M., Detken A., Bockmann A., & Meier B. H. (2003) NMR spectra of a microcrystalline protein at 30 kHz MAS. J. Am. Chem. Soc. 125, 15807–15810.
Chen L. L., et al. (2007) J-based 2D homonuclear and heteronuclear correlation in solid-state proteins. Magn. Reson. Chem. 45, S84-S92.
Chen L., et al. (2006) Constant-Time Through-Bond 13C Correlation Spectroscopy for Assigning Protein Resonances with Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 128, 9992–9993.
Chen L., et al. (2007) Backbone assignments in solid-state proteins using J-based 3D Heteronuclear correlation spectroscopy. J. Am. Chem. Soc. 129, 10650–10651.
Griffin R. G. (1998) Dipolar recoupling in MAS spectra of biological solids. Nat. Struct. Biol. 5 Suppl, 508–512.
Zhao X., Edén M., & Levitt M. H. (2001) Recoupling of heteronuclear dipolar interactions in solid-state NMR using symmetry-based pulse sequences. Chem. Phys. Lett. 342, 353–361.
Ladizhansky V. (2009) Homonuclear dipolar recoupling techniques for structure determination in uniformly 13C-labeled proteins. Solid State Nucl. Magn. Reson. 36, 119–128.
Balayssac S. p., Bertini I., Lelli M., Luchinat C., & Maletta M. (2007) Paramagnetic Ions Provide Structural Restraints in Solid-State NMR of Proteins. J. Am. Chem. Soc. 129, 2218–2219.
Nadaud P. S., Helmus J. J., Kall S. L., & Jaroniec C. P. (2009) Paramagnetic Ions Enable Tuning of Nuclear Relaxation Rates and Provide Long-Range Structural Restraints in Solid-State NMR of Proteins. J. Am. Chem. Soc. 131, 8108–8120.
Xu X., et al. (2009) Intermolecular dynamics studied by paramagnetic tagging. J. Biomol. NMR 43, 247–254.
Lian L.-Y. & Middleton D. A. (2001) Labelling approaches for protein structural studies by solution-state and solid-state NMR. Prog. Nucl. Magn. Reson. Spectrosc. 39, 171–190.
Schubert M., Manolikas T., Rogowski M., & Meier B. H. (2006) Solid-state NMR spectroscopy of 10% 13C labeled ubiquitin: spectral simplification and stereospecific assignment of isopropyl groups. J. Biomol. NMR 35, 167–173.
Delaglio F., et al. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293.
Mobli M., Maciejewski M. W., Gryk M. R., & Hoch J. C. (2007) Automatic maximum entropy spectral reconstruction in NMR. J. Biomol. NMR 39, 133–139.
Johnson B. A. & Blevins R. A. (1994) NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614.
Goddard T. D. & Kneller D. G. Sparky 3 (University of California, San Francisco).
Vranken W. F., et al. (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696.
Kraulis P. J. (1989) ANSIG: A program for the assignment of protein 1H 2D NMR spectra by interactive computer graphics. J. Magn. Reson. 84, 627–633.
Matsuki Y., Eddy M. T., & Herzfeld J. (2009) Spectroscopy by Integration of Frequency and Time Domain Information for Fast Acquisition of High-Resolution Dark Spectra. J. Am. Chem. Soc. 131, 4648–4656.
Laue E. D., Skilling J., Staunton J., Sibisi S., & Brereton R. G. (1985) Maximum Entropy Method in Nuclear Magnetic Resonance Spectroscopy. J. Magn. Reson. 62, 437–452.
Hoch J. C., Stern A. S., Donoho D. L., & Johnstone I. M. (1990) Maximum-Entropy Reconstruction of Complex (Phase-Sensitive) Spectra. J. Magn. Reson. 86, 236–246.
Barna J. C. J., Laue E. D., Mayger M. R., Skilling J., & Worrall S. J. P. (1987) Exponential Sampling, an Alternative Method for Sampling in Two-Dimensional NMR Experiments. J. Magn. Reson. 73, 69–77.
de Bouregas F. S. & Waugh J. S. (1992) ANTIOPE, a program for computer experiments on spin dynamics. J. Magn. Reson. 96, 280–289.
Smith S. A., Levante T. O., Meier B. H., & Ernst R. R. (1994) Computer Simulations in Magnetic Resonance. An Object-Oriented Programming Approach. J. Magn. Reson., Ser A 106, 75–105.
Blanton W. B. (2003) BlochLib: a fast NMR C++ tool kit. J. Magn. Reson. 162, 269–283.
Bak M., Rasmussen J. T., & Nielsen N. C. (2000) SIMPSON: A General Simulation Program for Solid-State NMR Spectroscopy. J. Magn. Reson. 147, 296–330.
Veshtort M. & Griffin R. G. (2006) SPINEVOLUTION: A powerful tool for the simulation of solid and liquid state NMR experiments. J. Magn. Reson. 178, 248–282.
Erickson-Viitanen S., et al. (1989) Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res. Hum. Retroviruses 5, 577–591.
Langsetmo K., Fuchs J., & Woodward C. (1989) Escherichia coli Thioredoxin Folds into 2 Compact Forms of Different Stability to Urea Denaturation. Biochemistry 28, 3211–3220.
Tasayco M. L. & Chao K. (1995) NMR study of the reconstitution of the beta-sheet of thioredoxin by fragment complementation. Proteins 22, 41–44.
Slaby I. & Holmgren A. (1975) Reconstitution of Escherichia coli thioredoxin from complementing peptide fragments obtained by cleavage at methionine-37 or arginine-73. J. Biol. Chem. 250, 1340–1347.
Sambrook J., Fritsch E. F., & Sambrook J. (1989) Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) 2nd Ed.
Byeon I. J., et al. (2009) Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function. Cell 139, 780–790.
Mossessova E. & Lima C. D. (2000) Ulp1-SUMO Crystal Structure and Genetic Analysis Reveal Conserved Interactions and a Regulatory Element Essential for Cell Growth in Yeast. Mol. Cell 5, 865–876.
Bennett A. E., Rienstra C. M., Auger M., Lakshmi K. V., & Griffin R. G. (1995) Heteronuclear Decoupling in Rotating Solids. J. Chem. Phys. 103, 6951–6958.
Chan J. C. C. & Tycko R. (2003) Recoupling of chemical shift anisotropies in solid-state NMR under high-speed magic-angle spinning and in uniformly 13C-labeled systems. J. Chem. Phys. 118, 8378–8389.
Wylie B. J., Franks W. T., & Rienstra C. M. (2006) Determinations of 15N Chemical Shift Anisotropy Magnitudes in a Uniformly 15N,13C-Labeled Microcrystalline Protein by Three-Dimensional Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy. J. Phys. Chem. B 110, 10926–10936.
Munowitz M., Aue W. P., & Griffin R. G. (1982) Two-dimensional separation of dipolar and scaled isotropic chemical shift interactions in magic angle NMR spectra. J. Chem. Phys. 77, 1686–1689.
Hohwy M., Jaroniec C. P., Reif B., Rienstra C. M., & Griffin R. G. (2000) Local structure and relaxation in solid-state NMR: Accurate measurement of amide N-H bond lengths and H-N-H bond angles. J. Am. Chem. Soc. 122, 3218–3219.
Hong M., Yao X., Jakes K., & Huster D. (2002) Investigation of Molecular Motions by Lee-Goldburg Cross-Polarization NMR Spectroscopy. J. Phys. Chem. B 106, 7355–7364.
van Rossum B. J., de Groot C. P., Ladizhansky V., Vega S., & de Groot H. J. M. (2000) A method for measuring heteronuclear (1H-13C) distances in high speed MAS NMR. J. Am. Chem. Soc. 122, 3465–3472.
Lorieau J. L. & McDermott A. E. (2006) Conformational flexibility of a microcrystalline globular protein: order parameters by solid-state NMR spectroscopy. J. Am. Chem. Soc. 128, 11505–11512.
Gullion T. & Schaefer J. (1989) Rotational-echo double-resonance NMR. J. Magn. Reson. 81, 196–200.
Bielecki A., Kolbert A. C., & Levitt M. H. (1989) Frequency-switched pulse sequences: Homonuclear decoupling and dilute spin NMR in solids. Chem. Phys. Lett. 155, 341–346.
Wickramasinghe N. P., Kotecha M., Samoson A., Past J., & Ishii Y. (2007) Sensitivity enhancement in (13)C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing (1)H T(1) relaxation. J. Magn. Reson. 184, 350–356.
Wickramasinghe N. P., et al. (2009) Nanomole-scale protein solid-state NMR by breaking intrinsic 1HT1 boundaries. Nat. Methods 6, 215–218.
Carravetta M., Edén M., Zhao X., Brinkmann A., & Levitt M. H. (2000) Symmetry principles for the design of radiofrequency pulse sequences in the nuclear magnetic resonance of rotating solids. Chem. Phys. Lett. 321, 205–215.
Holl S. M., McKay R. A., Gullion T., & Schaefer J. (1990) Rotational-echo triple-resonance NMR. J. Magn. Reson. 89, 620–626.
Gullion T., Baker D. B., & Conradi M. S. (1990) New, compensated Carr-Purcell sequences. J. Magn. Reson. 89, 479–484.
Vinogradov E., Madhu P. K., & Vega S. (1999) High-resolution proton solid-state NMR spectroscopy by phase-modulated Lee-Goldburg experiment. Chem. Phys. Lett. 314, 443–450.
Erickson-Viitanen S., Manfredi J., Viitanen P., Tribe D. E., Tritch R., Hutchison C. A., 3rd, Loeb D. D., & Swanstrom R. (1989) Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res. Hum. Retroviruses 5, 577–591.
Marley J., Lu M., & Bracken C. (2001) A method for efficient isotopic labeling of recombinant proteins. J. Biomol. NMR 20, 71–75.
Acknowledgments
The projects discussed here are supported by the National Institutes of General Medical Sciences (NIH Grants P50GM082251 and R01GM085306) and the National Center for Research Resources (NIH Grants P20RR017716-07 and P20RR015588). The authors thank Maria Luisa Tasayco, Dabeiba Marulanda, Jun Yang, Marcela Cataldi, Vilma Arriaran for their contributions to the preparation of thioredoxin reassemblies and/or solid-state NMR studies of these reassemblies.
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Sun, S. et al. (2012). Solid-State NMR Spectroscopy of Protein Complexes. In: Shekhtman, A., Burz, D. (eds) Protein NMR Techniques. Methods in Molecular Biology, vol 831. Humana Press. https://doi.org/10.1007/978-1-61779-480-3_17
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