**5. Examples of Raman assistance in chemical modifications of protein crystals**

Application of complementary techniques that probe different features is an added value to the understanding of the relationship between structure, function and dynamics.

Raman microscopy proved to be a valuable support to protein crystallography in all the steps of the 3D structure determination, from the preparation of the derivative crystals up to the interpretation of the electron density maps. We will first present examples of chemical composition changes (cfr 5.1), and then we will move to possible secondary (cfr. 5.2) and tertiary structure (cfr 5.3) modifications occurring upon a physico-chemical treatment.

In some cases crystallography can assist Raman spectroscopy in defining frequency assignments. In this case we speak about crystallography-assisted Raman spectroscopy.

### **5.1 Raman helping phase problem solution**

Phase problem in crystallography can be solved for new proteins *via* multiple wavelength anomalous diffraction (MAD) or by multiple isomorphous replacement (MIR). Both these approaches usually require a chemical modification of the protein crystals.

#### **5.1.1 Raman detection of Se-Met incorporation into protein crystals**

Se-Met derivatives are expressed for phase determination using multiwavelength anomalous dispersion (MAD) experiments (Cassetta et al., 1999). Se-Met inclusion is a widespread approach for MAD experiments since Met residues are present with an average occurrence of 1 per 59 residues (Hendrikson et al., 1990). Once crystals of a Se-Met derivative protein have been obtained, only then the absorption spectrum can be used to reveal the presence of selenium for determination of the crystal structure. Se-Met derivative crystals should be stored in a reducing environment (usually adding DTT) and diffraction experiments should be carried out using fresh crystals. It has been reported that two-week storage of Se-Met protein crystals can produce significant deterioration of the crystal diffraction power (Doublié, 1997). Since synchrotron beam-time is not always available immediately after growth of the crystals, a tool to check the Se-Met status in stored crystals is beneficial.

A Raman microscopy study was conducted on isomorphous wild-type and Se-Met crystals of the βγ-crystallin from *Geodia cydonium* (geodin) (Vergara et al., 2008). Geodin is a protein of unknown function, whose structural characterization could provide information on how homologous βγ-crystallin monomeric proteins evolved.

Table 1 summarizes the most relevant Raman bands observed in off-resonant Raman spectra for the SeMet-derivative crystals of geodin. Spectra at low frequency region (400-1200 cm-1) are reported in Figures 2. Usually, the Raman spectrum of a polypeptide is subdivided into three main regions of interest: 1) the range between 870–1150 cm−1, associated with the

Raman band can be assigned according to literature or basing on theoretical calculations, isotopic substitution or symmetry considerations (Long, 2002). But usually, the last operations do require experienced spectroscopists (see par 5.1.1) . The comparison of native protein crystal and chemically modified crystal can be performed *via* difference spectra in case of off-resonance spectrum or just *via* a simple comparison of the spectra for resonance

Application of complementary techniques that probe different features is an added value to

Raman microscopy proved to be a valuable support to protein crystallography in all the steps of the 3D structure determination, from the preparation of the derivative crystals up to the interpretation of the electron density maps. We will first present examples of chemical composition changes (cfr 5.1), and then we will move to possible secondary (cfr. 5.2) and tertiary structure (cfr 5.3) modifications occurring upon a physico-chemical treatment. In some cases crystallography can assist Raman spectroscopy in defining frequency assignments. In this case we speak about crystallography-assisted Raman spectroscopy.

Phase problem in crystallography can be solved for new proteins *via* multiple wavelength anomalous diffraction (MAD) or by multiple isomorphous replacement (MIR). Both these

Se-Met derivatives are expressed for phase determination using multiwavelength anomalous dispersion (MAD) experiments (Cassetta et al., 1999). Se-Met inclusion is a widespread approach for MAD experiments since Met residues are present with an average occurrence of 1 per 59 residues (Hendrikson et al., 1990). Once crystals of a Se-Met derivative protein have been obtained, only then the absorption spectrum can be used to reveal the presence of selenium for determination of the crystal structure. Se-Met derivative crystals should be stored in a reducing environment (usually adding DTT) and diffraction experiments should be carried out using fresh crystals. It has been reported that two-week storage of Se-Met protein crystals can produce significant deterioration of the crystal diffraction power (Doublié, 1997). Since synchrotron beam-time is not always available immediately after growth of the crystals, a tool to check the Se-Met status in stored crystals

A Raman microscopy study was conducted on isomorphous wild-type and Se-Met crystals of the βγ-crystallin from *Geodia cydonium* (geodin) (Vergara et al., 2008). Geodin is a protein of unknown function, whose structural characterization could provide information on how

Table 1 summarizes the most relevant Raman bands observed in off-resonant Raman spectra for the SeMet-derivative crystals of geodin. Spectra at low frequency region (400-1200 cm-1) are reported in Figures 2. Usually, the Raman spectrum of a polypeptide is subdivided into three main regions of interest: 1) the range between 870–1150 cm−1, associated with the

**5. Examples of Raman assistance in chemical modifications of protein** 

the understanding of the relationship between structure, function and dynamics.

approaches usually require a chemical modification of the protein crystals.

**5.1.1 Raman detection of Se-Met incorporation into protein crystals** 

Raman (Carey, 2006).

**5.1 Raman helping phase problem solution** 

homologous βγ-crystallin monomeric proteins evolved.

**crystals** 

is beneficial.

vibrations of the backbone Cα-C and Cα-N bonds; 2) the range between 1230–1350 cm−1, containing the amide III region vibrations, associated with normal modes of various combinations of the Cα-H and N-H deformations together with the Cα-C and Cα-N stretchings (Asher et al., 2001); 3) the range between 1630–1700 cm−1, associated with C=O stretching modes, defined as the amide I region (Ngarize et al., 2004). Furthermore, the lower and higher regions can also be informative: i) the conformation and detection of disulphide bridges can be investigated in the low frequency (500-540 cm-1) region (Kudryatsev et al., 1998); ii) hydrophobic interactions can be investigated by analysing the C-H stretching region (2800-3200 cm-1) (Chourpa et al., 2006), and the S-H stretching region 2550-2600 cm-1 can serve as a valuable probe of local dynamics (Thomas, 1999).

Fig. 2. Low frequency Raman spectra of the wild-type geodin crystals (WT), the Se-Met labelled crystals (Se), the mother liquor from which both kind of crystals grew up (ML). Signals attributed to the mother liquor are tagged by a star. The spectral resolution is 4 cm-1. (after Vergara et al., 2008).

The Raman spectra collected on the isomorphous crystals of wild-type and Se-Met geodin crystals reveal the same secondary structure features (Amide I and III). These findings suggest that the presence of the Se-Met does not alter the structure of geodin, as observed for most of the proteins. The main difference between the two spectra (excluding the slightly different intensity of mother liquor signals) is in one narrow region at low frequency. Indeed, the bands in the 570-600 cm-1 region are present only in the Se-Met geodin crystal, and not in the spectra of the mother liquor or in the wild-type geodin crystal (Table 1). This

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 401

glycosilation, as already done by comparing spectra of native and derivative proteins in

As previously stated, heavy atoms can be incorporated into protein crystals for the purposes of phasing. The simplest way to soak a heavy atom into protein crystals is to immerse the crystal straight into a drop containing the heavy atom at the final concentration for a soaking time ranging from 10 min up to several days. Different salts can be used for this purpose: successful experiences have been reported using K2PtCl4, KAu(CN)2, K2HgI4, UO2 (C2H3O2)2, HgCl2, K3UO2F5 and para-chloromercurybenzoic sulfate (PCMBS) (Boggon &

During this procedure, typical question are: did actually the ion soak into the crystal? Is the

Raman microscopy can help answering these questions without waiting for a Patterson map. Reports of Raman-based direct Ptincorporation and indirect Eu3+ soaking are available in literature (Carpenter et al., 2007). Indeed, the Pt-Cl stretching appears at 330 cm-1 that can be compared to the Trp band at 760 cm-1. From the time-evolution of the relative intensity

Authors have recently defined a protocol to follow Hg2+ binding to free Cys residues (unpublished results), that is dependent on the number of Cys residues involved in the

In some rare cases chemical modifications, such as pH change or Cys-Cys reduction, can significantly change even secondary structure. By taking advantage from the sensitivity of Amide I band to conformational variations, Thomas revealed a β-sheet to α-helix transition upon TCEP [tris(2-carboxyethyl)phosphine] addition to bovine insulin crystals (Zheng et al., 2004). Unfortunately, upon chemical reduction crystals do not diffract anymore, and no crystallographic counterpart is available. An analogous conformational transition occurred upon pH decrease of 5S subunit of transcarboxylate (Zheng et al., 2004), where modifications in the Amide III region (Mikhonin et al., 2006) suggested a significant modification into the crystal matrixes. Also hydration can affect deeply Amide I, and eventually secondary structure, as observed for a collagen-like polypeptide, for which Raman spectrum of the lyophilized powder showed very different Amide I frequencies

In particular, in this case, spectra of (PPG)10 powders are characterized by three distinct well-resolved amide I bands (1638, 1655, 1690 cm−1), that do not match the frequencies

The three amide I bands, and especially the well-resolved band at 1690 cm−1, do not match the frequencies reported by FT-IR solution studies for the unfolded (PPG)10 (maximum at 1633 and a shoulder at 1665 cm−1) or for the unfolded polyproline (one band at 1621 cm−1)

The amide I bands of (PPG)10 crystals and powders are significantly different, not only for the intensity distribution of the crystal spectrum (depending on the orientation of the polarization of the exciting laser beam with respect to the main axes of the Raman tensor in the respective

unit cell), but also for the frequency of the Raman peaks (After Merlino et al., 2008a).

**5.2 Raman detection of chemical modifications perturbing secondary structure** 

when compared to single crystals (Figure 3, from Merlino at al., 2008a).

observed in the spectra of (PPG)10 crystals (1629, 1645 and 1669 cm−1).

time of soaking long enough for allowing the heavy atom diffusion into the crystal?

solution (Sundararajan, et al. 2006; Brewster et al., 2011).

**5.1.2 Raman detection of heavy atom derivatives** 

I330/I760 we can follow Pt2+ soaking into the crystal.

Shapiro, 2000).

binding (1 or 2).

(Bryan et al., 2007).

spectral feature can be confidently assigned to the C-Se stretching, in agreement with previous Raman studies on selenium-containing organic compounds (Hamada & Morishima, 1977; Paetzold et al., 1967), and on the selenomethionine aminoacid (Zainal & Wolf, 1995; Lopez et al., 1981).


Table 1. Assignment of characteristic Raman bands measured for the Se-Met derivative crystals of the crystalline-like protein from *Geodia cydonium* (geodin).

Using these assignments, SeMet Raman peaks have been observed also in the SeMet derivative of protein SOUL (Rossi et al., 2009). In these crystals, a quantitative evaluation of the relative amount of SeMet replacement was also achieved by comparative analysis. In principle, Raman microspectra could be also used to reveal post translational modifications, such as phosphorylation, acetylation, trimethylation, ubiquitination and glycosilation, as already done by comparing spectra of native and derivative proteins in solution (Sundararajan, et al. 2006; Brewster et al., 2011).

### **5.1.2 Raman detection of heavy atom derivatives**

400 Current Trends in X-Ray Crystallography

spectral feature can be confidently assigned to the C-Se stretching, in agreement with previous Raman studies on selenium-containing organic compounds (Hamada & Morishima, 1977; Paetzold et al., 1967), and on the selenomethionine aminoacid (Zainal &

C=O stretching **Amide I** 

N-H, C-H and CH2 bending **Amide III** 

Ring stretching, side chain Tyr-Phe

C-C stretching Tyr-Phe

Fermi resonance doublet Tyr

Table 1. Assignment of characteristic Raman bands measured for the Se-Met derivative

Using these assignments, SeMet Raman peaks have been observed also in the SeMet derivative of protein SOUL (Rossi et al., 2009). In these crystals, a quantitative evaluation of the relative amount of SeMet replacement was also achieved by comparative analysis. In principle, Raman microspectra could be also used to reveal post translational modifications, such as phosphorylation, acetylation, trimethylation, ubiquitination and

**Primary Structure** 

**Se-Met** 

**Secondary structure** 

coil α-helix

α-helix random coil β sheet

β sheet and random

Wolf, 1995; Lopez et al., 1981).

1449 C-H2 scissoring

1127 C-C stretching 1100 C-C stretching

936 C-C, skeletal stretching

1400 COO-

**assignment of vibration mode** 

1577 Ring stretching Trp 1548 Ring stretching Trp

1032 C-C stretching Phe 1003 Ring breathing Phe

889 C-C, C-O deformations Trp

760 C-C, C-O deformations Trp 644 ring bending Tyr 622 ring bending Phe

> symmetric C-Se stretching asymmetric C-Se stretching

crystals of the crystalline-like protein from *Geodia cydonium* (geodin).

stretching

**Band frequency** 

1650 shoulder

**(cm-1)** 

1668

1614 1605

1318 1245 1237

1205 1159

855 829

598

578 shoulder

As previously stated, heavy atoms can be incorporated into protein crystals for the purposes of phasing. The simplest way to soak a heavy atom into protein crystals is to immerse the crystal straight into a drop containing the heavy atom at the final concentration for a soaking time ranging from 10 min up to several days. Different salts can be used for this purpose: successful experiences have been reported using K2PtCl4, KAu(CN)2, K2HgI4, UO2 (C2H3O2)2, HgCl2, K3UO2F5 and para-chloromercurybenzoic sulfate (PCMBS) (Boggon & Shapiro, 2000).

During this procedure, typical question are: did actually the ion soak into the crystal? Is the time of soaking long enough for allowing the heavy atom diffusion into the crystal?

Raman microscopy can help answering these questions without waiting for a Patterson map. Reports of Raman-based direct Ptincorporation and indirect Eu3+ soaking are available in literature (Carpenter et al., 2007). Indeed, the Pt-Cl stretching appears at 330 cm-1 that can be compared to the Trp band at 760 cm-1. From the time-evolution of the relative intensity I330/I760 we can follow Pt2+ soaking into the crystal.

Authors have recently defined a protocol to follow Hg2+ binding to free Cys residues (unpublished results), that is dependent on the number of Cys residues involved in the binding (1 or 2).

#### **5.2 Raman detection of chemical modifications perturbing secondary structure**

In some rare cases chemical modifications, such as pH change or Cys-Cys reduction, can significantly change even secondary structure. By taking advantage from the sensitivity of Amide I band to conformational variations, Thomas revealed a β-sheet to α-helix transition upon TCEP [tris(2-carboxyethyl)phosphine] addition to bovine insulin crystals (Zheng et al., 2004). Unfortunately, upon chemical reduction crystals do not diffract anymore, and no crystallographic counterpart is available. An analogous conformational transition occurred upon pH decrease of 5S subunit of transcarboxylate (Zheng et al., 2004), where modifications in the Amide III region (Mikhonin et al., 2006) suggested a significant modification into the crystal matrixes. Also hydration can affect deeply Amide I, and eventually secondary structure, as observed for a collagen-like polypeptide, for which Raman spectrum of the lyophilized powder showed very different Amide I frequencies when compared to single crystals (Figure 3, from Merlino at al., 2008a).

In particular, in this case, spectra of (PPG)10 powders are characterized by three distinct well-resolved amide I bands (1638, 1655, 1690 cm−1), that do not match the frequencies observed in the spectra of (PPG)10 crystals (1629, 1645 and 1669 cm−1).

The three amide I bands, and especially the well-resolved band at 1690 cm−1, do not match the frequencies reported by FT-IR solution studies for the unfolded (PPG)10 (maximum at 1633 and a shoulder at 1665 cm−1) or for the unfolded polyproline (one band at 1621 cm−1) (Bryan et al., 2007).

The amide I bands of (PPG)10 crystals and powders are significantly different, not only for the intensity distribution of the crystal spectrum (depending on the orientation of the polarization of the exciting laser beam with respect to the main axes of the Raman tensor in the respective unit cell), but also for the frequency of the Raman peaks (After Merlino et al., 2008a).

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 403

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

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

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

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

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

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 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

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

be even used as restrains during crystallographic model refinement.

the oxidation, coordination and spin state of the iron ion).

haemochrome state (bands at 1361, 1496 and 1587 cm-1, respectively).

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

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

entry, or metal center.

(Thomas, 1999).

(Figure 4).

al., 2010).

and 0.5 for strong H-bond donation of the OH).

Fig. 3. Medium frequency (A) and low frequency (B) Raman spectra of (PPG)10 powders, crystals, and mother liquor from which crystals grew up. In spectra (A) and (B) the signals attributed to the mother liquor are tagged by a star. Spectral resolution is 4 cm−1. After (Merlino et al. 2008a).

#### **5.3 Raman detection of chemical modification perturbing tertiary structure**

Certainly, the most common structural modifications are related to tertiary structure. The amino-acidic modification can be followed even by off-resonance Raman spectra, whereas modification at metal center can only be detected *via* resonance Raman spectra.

Fig. 3. Medium frequency (A) and low frequency (B) Raman spectra of (PPG)10 powders, crystals, and mother liquor from which crystals grew up. In spectra (A) and (B) the signals attributed to the mother liquor are tagged by a star. Spectral resolution is 4 cm−1. After

Certainly, the most common structural modifications are related to tertiary structure. The amino-acidic modification can be followed even by off-resonance Raman spectra, whereas

**5.3 Raman detection of chemical modification perturbing tertiary structure** 

modification at metal center can only be detected *via* resonance Raman spectra.

(Merlino et al. 2008a).
