**Complementary Use of NMR to X-Ray Crystallography for the Analysis of Protein Morphological Change in Solution**

Shin-ichi Tate, Aiko Imada and Noriaki Hiroguchi *Department of Mathematical and Life Sciences, Hiroshima University Japan* 

#### **1. Introduction**

408 Current Trends in X-Ray Crystallography

Rossi, B., G. Mariotto, E. Ambrosi & H. L. Monaco (2009) "Raman scattering investigation of

Smulevich, G. & Spiro, T. G. (1993) "Single-crystal micro-Raman spectroscopy" Meth.

Stoner-Ma, D., Skinner, J.M., Schneider, D.K., Cowan,M., Sweet, R.M. & Oliver, A.M., (2011)

Sundararajan, N. Mao, D. Q., Chan, S., Koo, T. W., Su, X., Sun, L., Zhang, J.W., Sung, K. B.,

Tardieu, A. (1998) "Alpha-crystallin quaternary structure and interactive properties control

Thomas, G. J. J. (1999) "Raman spectroscopy of protein and nucleic acid assemblies" Ann.

Tsuboi, M., Benevides, J.M. &. Thomas, G.J. Jr. (2007) "The Complex of Ethidium Bromide

Vergara, A., Lorber, B. Zagari, A., Giegé, R. (2003) "Physical aspects of protein crystal

Vergara, A., Merlino, A., Pizzo, E., D'Alessio, G. & Mazzarella, L. (2008) "A novel method

Vergara, A., Castagnolo, D., Carotenuto, L., Vitagliano, L., Berisio, R., Sorrentino, G., García-

Vergara, A., Vitagliano, L., Marino, K., Merlino, A., Sica, F., Verde, C., di Prisco G.

Vitagliano, L., Vergara A., Bonomi, G., Merlino, A., Smulevich, G., Howes, B., di Prisco, G.,

Zheng, R., Zheng, X., Dong, J. & Carey, P.R. (2004) "Proteins can convert to β-sheet in single

with Genomic DNA: Structure Analysis by Polarized Raman Spectroscopy"

growth investigated with the Advanced Protein Crystallization Facility in reduced

for detection of seleno-methionine incorporation in protein crystals *via* Raman

Ruiz, J.-M. , Zagari, A. (2009)"Phase behavior and crystallogenesis under counterdiffusion conditions of the collagen-model peptide (Pro-Pro-Gly)10" J. Cryst.

Mazzarella, L. (2010) "An order-disorder transition plays a role in switching off the

Verde, C. & L. Mazzarella, (2008) "Spectroscopic and crystallographic analysis of a tetrameric hemoglobin oxidation pathway reveals features of an intermediate R/T

eye lens transparency" Int. J. Biol. Macromol., 22, 211

gravity environments" Acta Cryst. D: Biol. Cryst., 59, 2

Root effect in fish hemoglobins" J. Biol. Chem., 285, 32568

40, 1844.

Enzymol., 226, 397

Anal. Chem., 78, 3543

Biophys. J., 92 928

Growth, 311, 304

of the NSLS" J. Synchr. Rad., 18, 37

Rev. Biophys. Biomol. Struct., 28, 1

microscopy" Acta Cryst. D., D64, 167

state" J. Am. Chem. Soc., 130, 10527

crystals" Prot. Sci., 13, 1288

selenomethionine replacement in protein SOUL crystals" J. Raman Spectroscopy,

"Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C

Yamakawa, M., Gafken, P. R., Randolph, T., McLerran, D., Feng, Z. D., Berlin, A. A. & Roth, M. B. (2006) "Ultrasensitive detection and characterization of posttranslational modifications using Surface-Enhanced Raman Spectroscopy"

> A vast amount of protein structure data is going to pave new ways in protein structure research. They improved the quality of predicted protein structure from its primary sequence (Sanchez and Sali 2000). The possible protein interaction sites to small ligand and/or the other proteins could be predicted based on the protein complex structures in the Protein Data Bank (PDB) (Morris et al. 2009). The combined use of bioinformatics with the protein structure data has been frequently giving invaluable outcomes to facilitate the understanding on the experimental results in biochemistry and molecular biology. *In silico* protein structure analyses are now already essential approaches in protein research.

> Protein structural data, most of which came from X-ray crystallography, are also useful to expand the protein structure analysis in solution, when combined with NMR. NMR chemical shift perturbation of a protein caused by the interaction with a compound, for example, allows sensitive identification of the interaction sites on protein (Shuker et al. 1996). This NMR derived binding site information with the protein structure facilitates drug design (Hajduk et al. 1997). This chemical shift-based approach is also applied to the protein-protein interaction, which enables to build a model protein complex structure (de Vries, van Dijk and Bonvin 2010, Dominguez, Boelens and Bonvin 2003). Although these approaches are now prevailingly used, there are limitations in their application. The approaches assume that the target protein retains the X-ray structure in solution and also negates the possible structural change caused by binding to a compound or a partner protein. Some of the proteins are known to have different domain arrangement from those in crystal (Skrynnikov et al. 2000a); it is often the case for the protein having domains linked by flexible linker. In addition, it is commonly found that proteins show structural change in response to compound binding or interaction with the other protein (Evenas et al. 2001). To expand the utility of protein structure data in the PDB in solution protein science, we need new NMR techniques to overcome the known limitations in the existing approaches, which could determine the structure changed from the one by X-ray in binding to a ligand or a partner protein to improve the modelled complex structure, for example.

> Protein structure change caused by interaction with other molecules is primarily important in discussing protein functional regulation. The structural change is not limited in the region around the binding site. Sometimes, it also causes rather global change including domain

Complementary Use of NMR to X-Ray Crystallography

**2. Residual anisotropic spin interactions** 

the clues to analyze protein morphology.

chemical shift anisotropy (CSA) (Fig. 2).

a NMR spectrum is called as a '*weak*' alignment (Bax 2003).

interaction. (b) 15N chemical shift anisotropy (CSA) effect.

protein works more vividly.

for the Analysis of Protein Morphological Change in Solution 411

Utsunomiya-Tate 2004). In this chapter, we are going to describe this NMR method from its theoretical backgrounds to applications to demonstrate how it works on the protein morphology analyses. This gives a complementary view on protein structure in solution, which is not seen by X-ray crystallography, although it requires high-resolution crystal structure as a template for the analysis. The combined use of high-resolution X-ray structure and its morphology analysis in solution by DIORITE may expand our understanding how

Conventional NMR structure analysis based on the short-range spin interactions does not give any global structural information required in protein morphology study. The residual anisotropic spin interactions, which become apparent for a '*weakly*' aligned protein, give molecular orientation information relative to the magnetic field. And the information gives

Two different types of residual anisotropic spin interactions are observed in the amide 1H-15N spin pair on a peptide plane; one is the nuclear spin dipolar-dipolar interaction and the other is anisotropic shielding effect against the external magnetic field, which is called as

These anisotropic spin interactions are not observed on a NMR spectrum for protein in an isotropic solution, where protein rapidly tumbles. Because of the rapid rotation that allows for protein to direct the entire angles against a magnetic field, anisotropic spin interactions are completely canceled for isotropic sample. In contrast, protein dissolved in a magnetically ordering liquid crystalline medium, for example, experiences some rotational restrictions by steric bump with the liquid crystalline molecules. Thus, it leads to incomplete cancellation of anisotropic spin interactions, which should give the residual anisotropic spin interactions observed on a spectrum. In a properly tuned liquid crystalline concentration, there is enough space to allow protein tumbling to some extent. The protein in an aligned liquid crystalline medium, accordingly, still can give high resolution NMR signals. In the higher liquid crystalline concentration, the protein tumbling becomes substantially limited and its NMR signals become severely broadened to prohibit their spectral observation. The condition that protein tumbling is slightly restricted to achieve incomplete cancellation of the anisotropic spin interactions, but it still gives narrow lines enough for the observation on

Fig. 2. Two anisotropic spin interactions observed for amide 1H-15N spin pair. (a) dipolar

rearrangement or subunit rearrangement. Hereafter, we would mention to this global structural change as '*protein morphological change*' to emphasize its large amplitude in motion. Protein morphological change is characterized by the considerable difference in the spatial arrangement of structural units from their original positions; structural units may include domain, subunit, or, sometimes, a small segment comprising of secondary structures. In the case, each unit mostly retains the original structure, except for the limited region engaged in the interaction (Fig. 1). The high-resolution unit structures from X-ray are therefore used as the templates in analyzing the morphological changes, for example, to see how the domains are rearranged upon ligand binding. As exemplified by Luciferase morphological change upon ligand binding, each domain shows little structural change, except for the intrinsically flexible loops (Nakatsu et al. 2006, Conti, Franks and Brick 1996) (Fig. 1). The bound form structure can be reconstituted from the apo-structure by rotating the small domain relative to the other (Fig. 1).

Fig. 1. Protein morphological change. Domain rearrangement of Luciferase upon binding to ATP. PDB codes 1LCI and 2D1Q for apo- and AMP-bound forms. Small domain is rotated over onto the large domain upon ligand binding. Both domains basically retain the structures in apo-form only with slight local changes.

The conventional NMR structure determination relying on NOEs and vicinal spin couplings, both of which give the only short-range structural information, does not properly determine the domain arrangement, particularly in the case that domains are connected by a linker and there are little inter-domain contacts. The accumulation of the less quantitative short-range structural information may even result in the erroneous determination of domain arrangement. It requires the global structural information that directly defines the domain arrangement.

In addition to the requirement for the global structural information, the molecular size limitation in solution NMR spectroscopy is another obstacle to work on the protein morphology by NMR. The conventional NMR approach has severe size limitation in determining protein structure, which is practically 30 kDa. Proteins having multiple domains tend to be greater than 50 kDa, thus the conventional NMR approach is not usable in protein morphology analysis.

Protein morphological change is of the great significance in biology, but there has not been any appropriate NMR techniques for this purpose, so far. With this concern, we have devised a new NMR approach, DIORITE (Determination of the Induced ORIentation by Trosy Experiments), that can overcome the obstacles in the conventional approaches, lacking global structural information and size limitation (Tate 2008, Tate, Shimahara and

rearrangement or subunit rearrangement. Hereafter, we would mention to this global structural change as '*protein morphological change*' to emphasize its large amplitude in motion. Protein morphological change is characterized by the considerable difference in the spatial arrangement of structural units from their original positions; structural units may include domain, subunit, or, sometimes, a small segment comprising of secondary structures. In the case, each unit mostly retains the original structure, except for the limited region engaged in the interaction (Fig. 1). The high-resolution unit structures from X-ray are therefore used as the templates in analyzing the morphological changes, for example, to see how the domains are rearranged upon ligand binding. As exemplified by Luciferase morphological change upon ligand binding, each domain shows little structural change, except for the intrinsically flexible loops (Nakatsu et al. 2006, Conti, Franks and Brick 1996) (Fig. 1). The bound form structure can be reconstituted from the apo-structure by rotating the small domain relative

Fig. 1. Protein morphological change. Domain rearrangement of Luciferase upon binding to ATP. PDB codes 1LCI and 2D1Q for apo- and AMP-bound forms. Small domain is rotated over onto the large domain upon ligand binding. Both domains basically retain the

The conventional NMR structure determination relying on NOEs and vicinal spin couplings, both of which give the only short-range structural information, does not properly determine the domain arrangement, particularly in the case that domains are connected by a linker and there are little inter-domain contacts. The accumulation of the less quantitative short-range structural information may even result in the erroneous determination of domain arrangement. It requires the global structural information that directly defines the

In addition to the requirement for the global structural information, the molecular size limitation in solution NMR spectroscopy is another obstacle to work on the protein morphology by NMR. The conventional NMR approach has severe size limitation in determining protein structure, which is practically 30 kDa. Proteins having multiple domains tend to be greater than 50 kDa, thus the conventional NMR approach is not usable

Protein morphological change is of the great significance in biology, but there has not been any appropriate NMR techniques for this purpose, so far. With this concern, we have devised a new NMR approach, DIORITE (Determination of the Induced ORIentation by Trosy Experiments), that can overcome the obstacles in the conventional approaches, lacking global structural information and size limitation (Tate 2008, Tate, Shimahara and

structures in apo-form only with slight local changes.

to the other (Fig. 1).

domain arrangement.

in protein morphology analysis.

Utsunomiya-Tate 2004). In this chapter, we are going to describe this NMR method from its theoretical backgrounds to applications to demonstrate how it works on the protein morphology analyses. This gives a complementary view on protein structure in solution, which is not seen by X-ray crystallography, although it requires high-resolution crystal structure as a template for the analysis. The combined use of high-resolution X-ray structure and its morphology analysis in solution by DIORITE may expand our understanding how protein works more vividly.
