**5.3.1 Off-resonance Raman spectra showing tertiary structure modifications**

Many Raman markers can provide information on the modified microenvironment of side chains (particularly free Cys and Tyr residues), disulfide bridges conformation or ligands entry, or metal center.

The relative intensity of Fermi resonance doublet from Tyr (I856/I830) is strictly dependent on the strength of the H-bond formed by tyrosines with the adjacent groups (1.25 exposed and 0.5 for strong H-bond donation of the OH).

Sulfidryl groups exposure of Cys residues can be investigate following the kinetics of H/D exchange *vs* temperature. Also the pKa of Cys can be extracted by titrating S-H stretching at 2550-70 cm-1 (mercaptoethanol 2580 cm-1) compared to the envelope at 2800-3000 cm-1 (Thomas, 1999).

When ligand soaking is followed, difference Raman spectra can provide indication on both bound ligands and related conformation changes of the proteins. Carey reported a review of Raman-microscopy applications to follow ligand binding (Carey, 2004), and more recently he has applied this technique in kinetic crystallography to monitor time evolution of βlactamases binding with clinical inhibitors (Carey, 2011). These spectroscopic evidences can be even used as restrains during crystallographic model refinement.

### **5.3.2 Resonance Raman spectra showing tertiary structure modifications**

Metal centers have been well studied *via* Resonance Raman (RR) spectroscopy for hemoprotein and not-containing heme proteins. A recent example was reported for the major haemoglobin from the sub-Antarctic fish *Eleginops maclovinus*, for which a variety of coordination, spin state were observed keeping the same oxidation states (Merlino et al., 2010). In this example, Raman microscopy experiments were conducted on two different carbomonoxy crystals (called Ortho and Hexa) as well as on their ferric and deoxy forms (Figure 4).

The high-frequency region (1300–1700 cm-1) of the RR spectrum includes the porphyrin inplane vibrational modes (which are sensitive to the electron density of the macrocycle and the oxidation, coordination and spin state of the iron ion).

The deoxy forms of both Ortho and Hexa are pentacoordinated high-spin states (bands at 1355, 1548–1549, 1582 and 1602–1607 cm-1). The ferric form contains a hexacoordinated lowspin hemichrome (bands at 1505, 1559, 1588 and 1640 cm-1) (Merlino et al., 2011). After long laser exposure times (about 10 min), the Ortho but not the Hexa form appears to be unstable under laser irradiation, and it irreversibly converts to a hexacoordinated low-spin haemochrome state (bands at 1361, 1496 and 1587 cm-1, respectively).

RR microscopy can be also used to identify anomaly in the coordination state of hemoprotein (Merlino et al., 2008b; Vergara et al., 2010). A recent and fine case is the unusual deoxy coordination found in the Antarctic fish hemoglobin from *Trematomus newnesi* (Hb1Tn) (Vergara et al., 2010). The crystal structure of deoxy form of this protein reveals distinct coordinations at the α and β hemes, and a high disorder at the EF helices of α heme, hosting the active site. Particularly, the distances His-Fe-His were unusual at the α subunits, raising doubt of hexa-coordination. The medium frequency region ruled out any contribution of bis-histidyl deoxy coordination, confirming a penta coordination. The low frequency regions clearly showed an heterogeneity in the Fe-His stretching, with a broad band attributed to the α/β structural differences in coordination (Figure 5, after Vergara et al., 2010).

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 405

Fig. 5. Resonance Raman spectra in the low-wavenumber region of Hb1Tn (major

*bernacchii*) and human HbA are reported (After Vergara et al., 2010).

**ray radiation damage** 

hemoglobin of *T. newnesi*) both in solution (solid lines) and in the crystal state (dashed lines) in the deoxy state. As reference of usual coordination also HbTb (major hemoglobin of *T.* 

**6. Raman-assisted biocrystallography to study chemical mechanisms and X-**

Raman assistance can go much beyond the crystal preparation and can provide additional structural information to solve some ambiguities in the electron density map interpretation. Examples include the 1) effect of X-ray damage as a function of the X-ray dose (Garman, 2010). Some examples are breakage of disulphide bonds (Carpentier et al., 2010) and Brominated-DNA photo-dissociation (McGeehan et al., 2007), and even X-ray-induced transient photobleaching in a photoactivatable green fluorescent Protein (Adam et al., 2009). Indeed, third generation synchrotrons brought back the problem of X-ray damage, even at 100 K (Garman, 2010). A complete microspectroscopic picture of the radiation damage is not yet available. The possibility to investigate the different rates of decay for different chemical groups would help crystallographers in making more stable derivative crystals. Authors are currently working on this subject in collaboration with SLS scientists. 2) X-ray induced photo-reduction that may affect metal spin and oxidation state. 3). Identification and freeze-

Fig. 4. Resonance Raman spectra of crystals of Hb1Em in 100 mM Tris–HCl buffer pH 8.0 at room temperature in the carbomonoxy (CO), deoxygenated (Deoxy) and ferric (Met) forms for Hexa (a) and Ortho (b) crystals. The Ortho deoxygenated form (b) converts quickly into haemochrome (Haemo) after 10 min laser exposure. Excitation wavelength, 514.5 nm; laser power at the sample 2 mW for the ferric and deoxy forms and 0.1 mW for the carbomonoxy form. All spectra were an average of at least six spectra with 2 min integration time (After Merlino et al., 2010).

Fig. 4. Resonance Raman spectra of crystals of Hb1Em in 100 mM Tris–HCl buffer pH 8.0 at room temperature in the carbomonoxy (CO), deoxygenated (Deoxy) and ferric (Met) forms for Hexa (a) and Ortho (b) crystals. The Ortho deoxygenated form (b) converts quickly into haemochrome (Haemo) after 10 min laser exposure. Excitation wavelength, 514.5 nm; laser power at the sample 2 mW for the ferric and deoxy forms and 0.1 mW for the carbomonoxy form. All spectra were an average of at least six spectra with 2 min integration time (After

Merlino et al., 2010).

Fig. 5. Resonance Raman spectra in the low-wavenumber region of Hb1Tn (major hemoglobin of *T. newnesi*) both in solution (solid lines) and in the crystal state (dashed lines) in the deoxy state. As reference of usual coordination also HbTb (major hemoglobin of *T. bernacchii*) and human HbA are reported (After Vergara et al., 2010).

#### **6. Raman-assisted biocrystallography to study chemical mechanisms and Xray radiation damage**

Raman assistance can go much beyond the crystal preparation and can provide additional structural information to solve some ambiguities in the electron density map interpretation. Examples include the 1) effect of X-ray damage as a function of the X-ray dose (Garman, 2010). Some examples are breakage of disulphide bonds (Carpentier et al., 2010) and Brominated-DNA photo-dissociation (McGeehan et al., 2007), and even X-ray-induced transient photobleaching in a photoactivatable green fluorescent Protein (Adam et al., 2009). Indeed, third generation synchrotrons brought back the problem of X-ray damage, even at 100 K (Garman, 2010). A complete microspectroscopic picture of the radiation damage is not yet available. The possibility to investigate the different rates of decay for different chemical groups would help crystallographers in making more stable derivative crystals. Authors are currently working on this subject in collaboration with SLS scientists. 2) X-ray induced photo-reduction that may affect metal spin and oxidation state. 3). Identification and freeze-

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 407

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Merlino, A., Verde, C., di Prisco, G., Mazzarella, L. & Vergara, A. (2008b)"Reduction of ferric

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Merlino, A. , Howes, B.D., di Prisco, G., Verde, C. , Smulevich, G. , Mazzarella, L. & Vergara,

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Raman and x-ray crystallography for the collagen-model peptide (Pro-Pro-Gly)10"

hemoglobin from Trematomus bernacchii in a partial bis-histidyl state produces a deoxy coordination even when encapsulated into the crystal phase". (2008b).

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Verde, C., di Prisco, G., Smulevich, G., Mazzarella, L. & Vergara, A. (2010) "Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the

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trapping of reaction intermediates (Carpenter et al., 2011). Bourgeois' group was pioneer in this field reporting non-resonance Raman of a trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase, a nonheme mononuclear iron enzyme that scavenges superoxide radicals (Katona et al., 2007), and further investigations came on RNA polymerase reactions as well (Carey et al., 2011). However this is out of the scope of this chapter, although it represents a formidable challenge for Raman-assisted X-ray biocrystallography.

### **7. Aknowledgements**

This work was financially supported by PNRA.

#### **8. References**


trapping of reaction intermediates (Carpenter et al., 2011). Bourgeois' group was pioneer in this field reporting non-resonance Raman of a trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase, a nonheme mononuclear iron enzyme that scavenges superoxide radicals (Katona et al., 2007), and further investigations came on RNA polymerase reactions as well (Carey et al., 2011). However this is out of the scope of this chapter, although it represents a formidable challenge for Raman-assisted X-ray

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Bourgeois, D. (2007) "Raman-assisted crystallography reveals end-on peroxide

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studies of collagen model peptides: complementary experimental and simulation

biocrystallography.

**8. References** 

**7. Aknowledgements** 

This work was financially supported by PNRA.


**18** 

*Japan* 

**Complementary Use of NMR to X-Ray** 

*Department of Mathematical and Life Sciences, Hiroshima University* 

**Morphological Change in Solution** 

Shin-ichi Tate, Aiko Imada and Noriaki Hiroguchi

**Crystallography for the Analysis of Protein** 

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 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

protein structure analyses are now already essential approaches in protein research.

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

**1. Introduction** 

