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Combination of NMR Spectroscopy and X-Ray Crystallography Offers Unique Advantages for Elucidation of the Structural Basis of Protein Complex Assembly

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Combination of NMR Spectroscopy and X-Ray Crystallography Offers Unique Advantages for Elucidation of the Structural Basis of Protein Complex Assembly SCIENCE CHINA Life Sciences • COVER ARTICLE • February 2011 Vol.54 No.2: 101–111 doi: 10.1007/s11427-011-4137-2 Combination of NMR spectroscopy and X-ray crystallography offers unique advantages for elucidation of the structural basis of protein complex assembly FENG Wei1,2, PAN LiFeng1 & ZHANG MingJie1* 1Division of Life Science, Molecular Neuroscience Center, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; 2National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Received May 7, 2010; accepted July 7, 2010 NMR spectroscopy and X-ray crystallography are two premium methods for determining the atomic structures of macro-biomolecular complexes. Each method has unique strengths and weaknesses. While the two techniques are highly com- plementary, they have generally been used separately to address the structure and functions of biomolecular complexes. In this review, we emphasize that the combination of NMR spectroscopy and X-ray crystallography offers unique power for elucidat- ing the structures of complicated protein assemblies. We demonstrate, using several recent examples from our own laboratory, that the exquisite sensitivity of NMR spectroscopy in detecting the conformational properties of individual atoms in proteins and their complexes, without any prior knowledge of conformation, is highly valuable for obtaining the high quality crystals necessary for structure determination by X-ray crystallography. Thus NMR spectroscopy, in addition to answering many unique structural biology questions that can be addressed specifically by that technique, can be exceedingly powerful in mod- ern structural biology when combined with other techniques including X-ray crystallography and cryo-electron microscopy. NMR spectroscopy, X-ray crystallography, structural biology, protein complex assembly Citation: Feng W, Pan L F, Zhang M J. Combination of NMR spectroscopy and X-ray crystallography offers unique advantages for elucidation of the struc- tural basis of protein complex assembly. Sci China Life Sci, 2011, 54: 101–111, doi: 10.1007/s11427-011-4137-2 Since the first protein structure was determined in the mid- crystallize, or even cannot be crystallized, because of fac- dle of last century, structural biology has had a critical role tors such as intrinsically disordered regions and the hetero- in driving the development of modern biology, chiefly by geneous conformational properties of samples. Conse- providing atomic level resolution of individual as well as quently, the most critical step for X-ray-based structure de- complexed forms of biological molecules. X-ray crystal- termination has been and will continue to be obtaining high lography and NMR spectroscopy are the two main methods quality crystals of samples of interest. By contrast, NMR that have been used for deriving high resolution atomic pic- spectroscopy can determine biomolecular structures in solu- tures of biomolecules, though cryo-electron microscopy is tion or in their solid state without the need to obtain crystals gaining momentum in this research direction. X-ray crys- [3–8]. NMR spectroscopy has been particularly useful for tallography was first developed to determine biomolecular defining the conformation of proteins with low molecular structures based on X-ray diffraction of single crystals [1,2] weights as well as proteins with large intrinsically disor- (Figure 1B). However, many biomolecules are difficult to dered regions [4,5,9–11]. Nonetheless, inherent technical deficiencies as well as practical limitations of NMR spec- troscopy limit the wider applications of the technique to *Corresponding author (email: [email protected]) © The Author(s) 2011. This article is published with open access at Springerlink.com life.scichina.com www.springer.com/scp 102 Feng W, et al. Sci China Life Sci February (2011) Vol.54 No.2 Figure 1 NMR spectroscopy and X-ray crystallography are two premium methods of structural biology. A, A typical 1H-15N HSQC spectrum of a protein. One cross peak in the spectrum (indicated by the arrow) represents one amino acid in the protein. B, An example X-ray diffraction map of a protein. The diffraction data can be converted into the electron density map of each amino acid in the protein. C, Understanding of the functions of biomolecules requires various information including their 3-D structures, detailed interactions, as well as dynamic properties. NMR spectroscopy can be used to determine the structures, dissect molecular interactions, and investigate the dynamics of proteins. X-ray crystallography is particularly powerful for determination of the atomic structures of highly complicated biological systems. determine high quality structures of proteins with large mo- emphasized that there is essentially no molecular size limit lecular weights. Thus, NMR spectroscopy and X-ray crys- for recording chemical shift information of proteins or their tallography are generally regarded as complementary tech- complexes by NMR spectroscopy. niques for the structure determination of biomolecules NMR spectroscopy has been highly successful in the de- [4,12,13]. termination of biomolecular structures [3,14–16]. As the Compared with X-ray crystallography, NMR spectros- chemical shift of each atom is highly sensitive to changes copy has unique features. The capability of NMR spectros- induced by environmental perturbations such as target copy for detecting individual atoms in terms of chemical binding, NMR spectroscopy has also been used to investi- shifts, coupled with the exquisite sensitivity of chemical gate biomolecular interactions since its first applications in shifts of atoms to their conformational environment, makes biology [17–19]. Furthermore, NMR spectroscopy is it the most powerful of all spectroscopic techniques. From uniquely suited for, and has been exceedingly powerful in the structural biology viewpoint, NMR spectroscopy is the investigating dynamic properties of biomolecules [20–24]. only method that allows direct monitoring of the conforma- The three-dimensional structure, interaction with its binding tional properties of a protein or its complex at the individual partners, and dynamic properties together provide a more amino acid level (e.g. via recording 1H, 15N and 13C chemi- complete understanding of cellular functions of a bio- cal shifts; Figure 1A), without any prior conformational molecule (Figure 1C). NMR spectroscopy is probably the knowledge of the protein. In addition, NMR spectroscopy only technique that alone can provide atomic details in all allows monitoring of conformational changes of a protein or three of these areas, and thus is recognized as an indispen- its complex upon the introduction of changes to the sample sable method in modern structural biology [25]. Because of condition. These two features of NMR spectroscopy render space limitations, we limit this review to the combination of the technology exceedingly powerful when compared with NMR spectroscopy and X-ray crystallography for the study X-ray crystallography, as crystallization processes are of the structure and function of proteins and protein com- largely based on trial-and-error methods. Finally, recent plex assemblies. developments of NMR technology (cryogenic detection NMR spectroscopy and X-ray crystallography are highly system combined with ultra-high magnetic field) have es- complementary in terms of structural determinations, and in sentially removed the major limitation of the technique the past have been generally used separately to investigate which is its intrinsically low sensitivity. It is now possible biomolecular complexes. Recently, NMR spectroscopy has to record a good quality NMR spectrum (e.g. 1H-15N HSQC been frequently combined with X-ray crystallography to spectrum) of a protein sample at a concentration as low as dissect the structures of complicated biomolecular com- 10 μmol L−1 within a few hours. Moreover, it should be plexes. The sensitivity and ultra-high resolution of NMR Feng W, et al. Sci China Life Sci February (2011) Vol.54 No.2 103 chemical shifts make NMR spectroscopy a unique tool for the isolated L27 domains demonstrated that neither of them investigating conformational properties of biomolecules at forms a defined conformation, as indicated by severe peak the atomic level without any prior structural information broadening or the narrowly dispersed peaks that are indica- (Figure 1A). More importantly, the quality of the NMR tive of unfolded proteins (Figures 2C1 and C2). Interest- 15 spectrum (i.e. peak dispersion pattern, line-widths, and ho- ingly, when we mixed N-labelled L27SAP97 with unlabelled mogeneity) is directly related to the conformational prop- L27NmLIN2 or vice versa, sharp and well-dispersed peaks erty of the sample being investigated. Such NMR-derived appeared, indicating that the L27 domain complex (con- information can be very valuable for obtaining high quality taining two related L27 domains) forms a more compact crystals of the molecules of interest. NMR spectroscopy can structural unit (Figures 2C3 and C4). Thus NMR spectros- be used for sample conditioning, and tracing changes in copy was used to detect the specific binding of a pair of protein behavior upon the addition of additives, and thus cognate L27 domains in solution, and to monitor conforma- nicely fills the gap in the “all-or-nothing” mode of crystal- tional transitions of the
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