**3. Achieving weak alignment**

In applying the residual anisotropic spin interactions described above, it is required to make a protein in a weakly aligned state. The aligning protein has to be carefully tuned to make the anisotropic interactions observable in a spectrum with keeping the spectral resolution and intensity. Alignment order is practically tuned to approximately 10-3, giving about 20 Hz in maximum absolute magnitude for amide 1H-15N RDC. To achieve a weak alignment, some artificial medium has to be used, because the inherent magnetic susceptibility of a globular protein is too small to align to the desired extent, except for some heme-containing proteins having substantial magnetic susceptibility associated with a heme group. In this section, we will review some media for weak alignment.

## **3.1 Magnetically aligning liquid crystalline media**

Magnetically ordering liquid crystalline media are commonly used. Discoidal phospholipid assembly, bicelle, is one of the prevailingly used materials for a weak alignment of protein (Ottiger and Bax 1999, Ottiger, Delaglio and Bax 1998, Tjandra and Bax 1997).. The bicelle is composed of a mixer of dimyristoylphosphatidylcholin (DMPC) and dihexynoylphosphatidylcholine (DHPC) in a ratio of 3:1. This phospholipid binary mixture forms lipid bilayers disks 30 nm – 40 nm in diameter. Bicelle has substantial magnetic susceptibility, and it spontaneously aligns under magnetic field with the normal of the bicelle surface staying perpendicular to the magnetic field (Fig. 6a).

In the experiments to measure the anisotropic spin interactions, an appropriate amount of bicelle is put into protein solution. In a high magnetic field, bicelles align and the aligned

Complementary Use of NMR to X-Ray Crystallography

spontaneously align in a high magnetic field.

through the electrostatic interaction not collisional interaction.

concentration of the phage suspension in protein solution.

increased ratio of bis-acrylamide generates smaller cavity.

two preparations change theprotein orientation to each other.

**3.3 Compressed acryl amide gel** 

to the cavities in the gel.

for the Analysis of Protein Morphological Change in Solution 419

Some naturally available molecules that spontaneously align in a magnetic field are also used for weak alignment. Purple membrane is one example, which forms two-dimensional crystal lattice structure of bacteriorhodopsin (bR) that is rich in -helices (Fig. 6b). Purple membrane constitutes of ordered -helices and thus it has intrinsic high anisotropic magnetic susceptibility. Because of the structural characteristics, purple membrane can

Suspension of purple membrane is added into protein solution to achieve a weak protein alignment. The alignment magnitude is tuned by the purple membrane concentration in a sample solution, as was the case for the bicelle medium (Sass et al. 1999). Purple membrane is the discoidal protein lipid complex, and it aligns with its normal parallel to the magnetic field. Purple membrane is rich in negative charge on its surface. Therefore, protein is aligned

Filamentous phage, which is made of a rod-like coat protein, is another example of the naturally occurring molecule used for weak alignment. Because of the highly anisotropic shape of the filamentous phage, it spontaneously aligns in a high magnetic field, with its rod axis parallel to a magnetic field (Fig. 6c). Filamentous phage has also negatively charged surface, thus it induces protein alignment through electrostatic interactions with protein as in the case of purple membrane. The alignment order can be tuned by adjusting the

The use of purple membrane and filamentous phage cannot be applied to basic proteins that are positively charged. The basic proteins tightly adsorb onto the negatively charged surfaces of the aligning molecules through the electrostatic interaction, which result in extreme ordering of proteins and thus prohibit observing high resolution NMR signals.

The other method uses anisotropically compressed acrylamide gel (Tycko, Blanco and Ishii 2000, Ishii, Markus and Tycko 2001, Sass et al. 1999). Acrylamide hydrogel has cavities to capture proteins within, whose cavity size is changed by alteringthe ratios of the composing chemicals, acrylamide and bis-acrylamide. Acrylamide forms a linear polymer chain and bis-acrylamide makes bridge to link acrylamide liner polymers, thus, the

The acrylamide gel has a spatially isotropic cavity. Protein in the gel does not show any preferential orientation, and thus it shows no residual anisotropic spin interaction on its NMR spectrum. Protein alignment by the gel is achieved by inducing the spatial anisotropy

In a weakly alignment experiment using the gel, a cast gel including a protein solution is inserted into a NMR sample tube. There are two ways to make anisotropically compressed gel; one is to press the gel along the NMR tube (compressed gel) and the other is squeeze it

In vertical compression, gel is cast to have a shorter diameter than the inner diameter for NMR sample tube. After inserting the gel, vertically compressed with glass rod in the tube (Fig. 7a). On the other hand, stretched gel is made from the cast gel having a slightly larger diameter than that the inner diameter of the tube; the gel is inserted into the tube by using tapered device (Chou et al. 2001) (Fig. 7b). Because of the different pressing direction, these

in the lateral dimension, thus it becomes stretched vertically (stretched gel) (Fig. 7).

**3.2 Naturally occurring materials that spontaneously align in a magnetic field** 

discoidal liquid crystalline molecules limit the space for protein tumbling. Bicelle has flat surface, thus, the protein involved in the aligned bicellar media will be surrounded by flat walls. Because of the steric clash, protein does not rotate freely near the bicelle wall, which will hider some orientations of the protein. This hindrance on some orientations for the protein in the bicellar medium causes incomplete rotational averaging of the anisotropic interactions, which thus makes the residual anisotropic interactions observables (Berlin,

O'Leary and Fushman 2009, Vijayan and Zweckstetter 2005, Zweckstetter 2008).

The aligning magnitude is readily tuned by the bicelle concentration; higher bicelle concentration induces the stronger order of alignment. The bicelle made of the binary phospholipid mixture involving DMPC and DHPC has neutral charge on its surface. Therefore, most proteins do not stick to bicelles. Protein aligns through collisional interaction described above.

Surface charge doping to the bicellar surface is possible. Incorporation of CTAB, cetyl trimethyl ammonium bromide, to DMPC/DHPC binary phospholipid bicelle generates the positively charged surface, while the addition of SDS, sodium dodecyl sulphate, makes it negatively charged. The surface charge doping to bicelle changes the aligning property from that by neutral bicelle. In charged bicellar solutoin, electrostatic interactions between the medium and protein become apparent. This makes it sometimes difficult to use the charged bicelle to acidic or basic proteins such as nucleic acid binding proteins.

There are some limitations in the bicelle application as aligning media. One is in the limited temperature range to keep bicelle in a liquid crystalline phase; it is typically 27°C – 45°C. Some proteins precipitate in this temperature range, and the bicelle is not used for such proteins. In addition, the bicelle is only stable around neutral pH; in acidic or basic solution, the bicellar medium tends to make phase-separation and loses aligning ability. To avoid the limitations associated with the physicochemical properties of the bicelle medium, other liquid crystalline media have been reported. By properly selecting the medium, we can now achieve weak alignment for rather various types of proteins, each of which requires its own optimal temperature, pH, and ionic strength (Prestegard, Bougault and Kishore 2004).

Fig. 6. Weak alignment made by using various media. (a) Discoidal shape phospholipid bicelle medium, (b) rod-like filamentous phage and (c) Purple membrane. Because of significant anisotropic magnetic susceptibility, the molecules spontaneously align in a magnetic field. Proteins within the aligning media are aligned through the collisional interaction or electrostatic interactions with the aligning molecules.

discoidal liquid crystalline molecules limit the space for protein tumbling. Bicelle has flat surface, thus, the protein involved in the aligned bicellar media will be surrounded by flat walls. Because of the steric clash, protein does not rotate freely near the bicelle wall, which will hider some orientations of the protein. This hindrance on some orientations for the protein in the bicellar medium causes incomplete rotational averaging of the anisotropic interactions, which thus makes the residual anisotropic interactions observables (Berlin,

The aligning magnitude is readily tuned by the bicelle concentration; higher bicelle concentration induces the stronger order of alignment. The bicelle made of the binary phospholipid mixture involving DMPC and DHPC has neutral charge on its surface. Therefore, most proteins do not stick to bicelles. Protein aligns through collisional

Surface charge doping to the bicellar surface is possible. Incorporation of CTAB, cetyl trimethyl ammonium bromide, to DMPC/DHPC binary phospholipid bicelle generates the positively charged surface, while the addition of SDS, sodium dodecyl sulphate, makes it negatively charged. The surface charge doping to bicelle changes the aligning property from that by neutral bicelle. In charged bicellar solutoin, electrostatic interactions between the medium and protein become apparent. This makes it sometimes difficult to use the charged

There are some limitations in the bicelle application as aligning media. One is in the limited temperature range to keep bicelle in a liquid crystalline phase; it is typically 27°C – 45°C. Some proteins precipitate in this temperature range, and the bicelle is not used for such proteins. In addition, the bicelle is only stable around neutral pH; in acidic or basic solution, the bicellar medium tends to make phase-separation and loses aligning ability. To avoid the limitations associated with the physicochemical properties of the bicelle medium, other liquid crystalline media have been reported. By properly selecting the medium, we can now achieve weak alignment for rather various types of proteins, each of which requires its own optimal temperature, pH, and ionic strength (Prestegard, Bougault and Kishore 2004).

Fig. 6. Weak alignment made by using various media. (a) Discoidal shape phospholipid bicelle medium, (b) rod-like filamentous phage and (c) Purple membrane. Because of significant anisotropic magnetic susceptibility, the molecules spontaneously align in a magnetic field. Proteins within the aligning media are aligned through the collisional

interaction or electrostatic interactions with the aligning molecules.

O'Leary and Fushman 2009, Vijayan and Zweckstetter 2005, Zweckstetter 2008).

bicelle to acidic or basic proteins such as nucleic acid binding proteins.

interaction described above.

#### **3.2 Naturally occurring materials that spontaneously align in a magnetic field**

Some naturally available molecules that spontaneously align in a magnetic field are also used for weak alignment. Purple membrane is one example, which forms two-dimensional crystal lattice structure of bacteriorhodopsin (bR) that is rich in -helices (Fig. 6b). Purple membrane constitutes of ordered -helices and thus it has intrinsic high anisotropic magnetic susceptibility. Because of the structural characteristics, purple membrane can spontaneously align in a high magnetic field.

Suspension of purple membrane is added into protein solution to achieve a weak protein alignment. The alignment magnitude is tuned by the purple membrane concentration in a sample solution, as was the case for the bicelle medium (Sass et al. 1999). Purple membrane is the discoidal protein lipid complex, and it aligns with its normal parallel to the magnetic field. Purple membrane is rich in negative charge on its surface. Therefore, protein is aligned through the electrostatic interaction not collisional interaction.

Filamentous phage, which is made of a rod-like coat protein, is another example of the naturally occurring molecule used for weak alignment. Because of the highly anisotropic shape of the filamentous phage, it spontaneously aligns in a high magnetic field, with its rod axis parallel to a magnetic field (Fig. 6c). Filamentous phage has also negatively charged surface, thus it induces protein alignment through electrostatic interactions with protein as in the case of purple membrane. The alignment order can be tuned by adjusting the concentration of the phage suspension in protein solution.

The use of purple membrane and filamentous phage cannot be applied to basic proteins that are positively charged. The basic proteins tightly adsorb onto the negatively charged surfaces of the aligning molecules through the electrostatic interaction, which result in extreme ordering of proteins and thus prohibit observing high resolution NMR signals.

#### **3.3 Compressed acryl amide gel**

The other method uses anisotropically compressed acrylamide gel (Tycko, Blanco and Ishii 2000, Ishii, Markus and Tycko 2001, Sass et al. 1999). Acrylamide hydrogel has cavities to capture proteins within, whose cavity size is changed by alteringthe ratios of the composing chemicals, acrylamide and bis-acrylamide. Acrylamide forms a linear polymer chain and bis-acrylamide makes bridge to link acrylamide liner polymers, thus, the increased ratio of bis-acrylamide generates smaller cavity.

The acrylamide gel has a spatially isotropic cavity. Protein in the gel does not show any preferential orientation, and thus it shows no residual anisotropic spin interaction on its NMR spectrum. Protein alignment by the gel is achieved by inducing the spatial anisotropy to the cavities in the gel.

In a weakly alignment experiment using the gel, a cast gel including a protein solution is inserted into a NMR sample tube. There are two ways to make anisotropically compressed gel; one is to press the gel along the NMR tube (compressed gel) and the other is squeeze it in the lateral dimension, thus it becomes stretched vertically (stretched gel) (Fig. 7).

In vertical compression, gel is cast to have a shorter diameter than the inner diameter for NMR sample tube. After inserting the gel, vertically compressed with glass rod in the tube (Fig. 7a). On the other hand, stretched gel is made from the cast gel having a slightly larger diameter than that the inner diameter of the tube; the gel is inserted into the tube by using tapered device (Chou et al. 2001) (Fig. 7b). Because of the different pressing direction, these two preparations change theprotein orientation to each other.

Complementary Use of NMR to X-Ray Crystallography

molecular shape of protein in the bulk solution.

chemical shifts should change from those found in an isotropic state.

see if any structure change happens through the interaction with the gel.

**4. RDC-based domain orientation analysis – Basics and limitation** 

emphasize the necessity of our TROSY based DIORITE approach that follows.

protein in the media, even in the cases of multiple-domain proteins.

that the bicelle induces a structural change to the protein.

changes caused by the interaction with the medium.

**4.1 Collecting the RDC data** 

for the Analysis of Protein Morphological Change in Solution 421

The mechanisms for aligning protein in magnetically aligned bicelle solution and rod-like filamentous phage suspension are well understood. The observed RDCs can be reproduced with using the protein crystal structure by the simulation based on the mechanism. The successful reproduction of the RDCs for the protein aligned by bicelle or filamentous phage supports that the proposed theory properly describes the state of protein in the aligning media. According to the theory, the observed residual anisotropic spin interactions can be established by a small fraction of protein experiencing the rotational restriction through the collision between the protein and the aligning molecule. In using the charged filamentous phage, the electrostatic interactions are also active in inducing the rotational restriction on the protein near the medium. The fraction for that is around 0.1% of the total number of protein molecules in the sample. The interactions have to be weak and transient. The intermolecule interactions, therefore, do not actively align the protein but just prohibit some fraction of the protein orientation near the medium. Under the condition, the aligning media do not induce artificial protein conformation or domain arrangement, because the prohibited orientation angles neat the medium is solely determined by the intrinsic

The structural perturbation by the medium is easily monitored by the NMR spectral changes. If the medium tightly interacts with protein to change the structure, some of the

The bicellar medium is in the liquid crystalline state over its phase-transition temperature. Under the temperature, it becomes a micelle solution that does not align in a magnetic field, thus does not induce protein alignment. If protein in this micelle solution does not show apparent spectral changes from the solution, including no micelle, we exclude the possibility

In the case of the charged filamentous phage, the increased concentration of cations, which shield the surface charge on the medium, impairs the ordering of the media in a magnetic field. The magnitudes of the anisotropic spin interactions for the protein in the phage suspension, therefore, are proportion to the cation concentration. If the spectral changes show linear dependency on the cation concentration, we could rule out the structural

Using compressed acrylamide gel allows the direct spectral comparison between the samples in the reference gel (non-compressed gel) and the isotropic solution without gel to

In general, under weakly aligning conditions, the spectral changes caused by the interaction with the media are rather small, ensuring that no apparent structural changes happen to

In this section, we will describe the experimental procedure to determine the domain orientation of a multiple-domain protein, from the RDC data collection to structure determination. In addition, the limitations in the RDC based analysis will be discussed to

RDC is measured on a pair of 1H coupled HSQC spectra for the samples in isotropic and anisotropic states. The 1H coupled HSQC spectrum gives a pair of split peaks along 15N axis

The charge doping in the anisotropically compressed gel is also possible. Negative charge is doped by replacing a part of acrylamide with acrylate, while a positive charge is introduced by adding DADMAC, diallyldimethylammonium chloride, in casting gel chip. These charge doping changes aligning property, because electrostatic interaction between protein and the gel becomes active.

Acrylamide gel is neutral and thus it induces protein alignment through collisional interaction between protein and walls having spatial anisotropy made by the compression. The non-charged acrylamide gel is readily used for any types of protein, independent on its pKa value. On the other hand, the charged gel is sometimes troublesome in achieving weak alignment of basic or acidic proteins, due to their adsorption onto the gel.

The aligning order magnitude is also tuned by changing the concentration of acrylamide and/or the ratio of the composing chemicals and also the diameter of the cased gel chip. In addition, the extent of the charge doping may have to be considered in some cases. There are more parameters to adjust than that for the liquid crystalline media. We, therefore, need some trial experiments to gain the optimal gel condition to have the anisotropic spin interactions observable in enough spectral resolution.

The great advantage in using the compressed gel is in the chemical stability of acrylamide gel. In using acrylamide gel for a weak alignment, we do not worry about the sample conditions including temperature, salt concentration, and solution pH. Under any sample conditions, we can align protein by the compressed gel. It is a keen difference from the limitation in applying liquid crystalline media and also in using the naturally occurring materials. The compressed gel is used as a universal media for weakly aligning protein.

Fig. 7. Anisotropically compressed gels to achieve weak alignment. (a) Vertically compressed gel. (b) Laterally compressed, thus, stretched gel. Cast gel chip (blue) is inserted into a NMR glass tube. In the vertical compression, the cast gel is pressed by a glass rod in NMR tube. These compressions induce spatially anisotropic cavities within the acrylamide gel.

#### **3.4 Protein structure in aligned media – Is it natural?**

A weak alignment of protein is prerequisite for the analysis of protein morphology by NMR, because it relies on the anisotropic spin interactions that happen only under a weak aligned condition. Because protein is dissolved in an artificial medium like magnetically ordering liquid crystalline solution, one should worry about the structure under the condition; the domain orientation in such a medium represents the real state of the protein in an isotropic solution?

The charge doping in the anisotropically compressed gel is also possible. Negative charge is doped by replacing a part of acrylamide with acrylate, while a positive charge is introduced by adding DADMAC, diallyldimethylammonium chloride, in casting gel chip. These charge doping changes aligning property, because electrostatic interaction between protein and the

Acrylamide gel is neutral and thus it induces protein alignment through collisional interaction between protein and walls having spatial anisotropy made by the compression. The non-charged acrylamide gel is readily used for any types of protein, independent on its pKa value. On the other hand, the charged gel is sometimes troublesome in achieving weak

The aligning order magnitude is also tuned by changing the concentration of acrylamide and/or the ratio of the composing chemicals and also the diameter of the cased gel chip. In addition, the extent of the charge doping may have to be considered in some cases. There are more parameters to adjust than that for the liquid crystalline media. We, therefore, need some trial experiments to gain the optimal gel condition to have the anisotropic spin

The great advantage in using the compressed gel is in the chemical stability of acrylamide gel. In using acrylamide gel for a weak alignment, we do not worry about the sample conditions including temperature, salt concentration, and solution pH. Under any sample conditions, we can align protein by the compressed gel. It is a keen difference from the limitation in applying liquid crystalline media and also in using the naturally occurring materials. The compressed gel is used as a universal media for weakly aligning protein.

Fig. 7. Anisotropically compressed gels to achieve weak alignment. (a) Vertically compressed gel. (b) Laterally compressed, thus, stretched gel. Cast gel chip (blue) is inserted into a NMR glass tube. In the vertical compression, the cast gel is pressed by a glass rod in NMR tube. These compressions induce spatially anisotropic cavities within the acrylamide gel.

A weak alignment of protein is prerequisite for the analysis of protein morphology by NMR, because it relies on the anisotropic spin interactions that happen only under a weak aligned condition. Because protein is dissolved in an artificial medium like magnetically ordering liquid crystalline solution, one should worry about the structure under the condition; the domain orientation in such a medium represents the real state of the protein in an isotropic

alignment of basic or acidic proteins, due to their adsorption onto the gel.

interactions observable in enough spectral resolution.

**3.4 Protein structure in aligned media – Is it natural?** 

solution?

gel becomes active.

The mechanisms for aligning protein in magnetically aligned bicelle solution and rod-like filamentous phage suspension are well understood. The observed RDCs can be reproduced with using the protein crystal structure by the simulation based on the mechanism. The successful reproduction of the RDCs for the protein aligned by bicelle or filamentous phage supports that the proposed theory properly describes the state of protein in the aligning media.

According to the theory, the observed residual anisotropic spin interactions can be established by a small fraction of protein experiencing the rotational restriction through the collision between the protein and the aligning molecule. In using the charged filamentous phage, the electrostatic interactions are also active in inducing the rotational restriction on the protein near the medium. The fraction for that is around 0.1% of the total number of protein molecules in the sample. The interactions have to be weak and transient. The intermolecule interactions, therefore, do not actively align the protein but just prohibit some fraction of the protein orientation near the medium. Under the condition, the aligning media do not induce artificial protein conformation or domain arrangement, because the prohibited orientation angles neat the medium is solely determined by the intrinsic molecular shape of protein in the bulk solution.

The structural perturbation by the medium is easily monitored by the NMR spectral changes. If the medium tightly interacts with protein to change the structure, some of the chemical shifts should change from those found in an isotropic state.

The bicellar medium is in the liquid crystalline state over its phase-transition temperature. Under the temperature, it becomes a micelle solution that does not align in a magnetic field, thus does not induce protein alignment. If protein in this micelle solution does not show apparent spectral changes from the solution, including no micelle, we exclude the possibility that the bicelle induces a structural change to the protein.

In the case of the charged filamentous phage, the increased concentration of cations, which shield the surface charge on the medium, impairs the ordering of the media in a magnetic field. The magnitudes of the anisotropic spin interactions for the protein in the phage suspension, therefore, are proportion to the cation concentration. If the spectral changes show linear dependency on the cation concentration, we could rule out the structural changes caused by the interaction with the medium.

Using compressed acrylamide gel allows the direct spectral comparison between the samples in the reference gel (non-compressed gel) and the isotropic solution without gel to see if any structure change happens through the interaction with the gel.

In general, under weakly aligning conditions, the spectral changes caused by the interaction with the media are rather small, ensuring that no apparent structural changes happen to protein in the media, even in the cases of multiple-domain proteins.
