**3. Handling of protein crystals for Raman microscopy experiments**

Raman microscopy is ideal for the monitoring of bioprocesses as it is non-destructive, inexpensive, rapid and quantitative. Its confocal nature makes it possible to focus through transparent capillary or directly on crystals kept in their crystallization reactor allowing straight analysis on the sample.

In co-crystallization experiments two independent Raman spectra are collected on native and derivative crystals, using the same mother liquor with the exception of the reactant. In this case, the comparative analysis of the two spectra can provide differences in a) number, b) position and c) intensity of protein Raman bands. Manipulation of crystals for Raman measurements requires standard handling.

### **3.1 Crystal sampling**

394 Current Trends in X-Ray Crystallography

plastic support above the surface of the reservoir (*sitting drop*). *Batch crystallization* is a method where the sample is mixed with precipitant and additives creating a homogenous crystallization medium. In the *free interface diffusion* the protein sample is stratified over the precipitant solution; over time the sample and precipitant diffuse into one another and crystallization may occur at the interface. In a *microdialysis experiment* the protein solution is equilibrated through a membrane against the precipitant solution over time in a stepwise manner. In some cases, to improve the X-ray diffraction properties the crystallization is performed in a gel medium. Agarose, agar and silica have been successfully used as gel materials to obtain protein crystals (Chayen, 1998; Vergara et al., 2003; Vergara et al., 2009). It is a frequent task to produce modified protein crystals in order to study the structural modifications undergoing a chemical treatment. These derivative crystals can be prepared *via* co-crystallization, or *via* soaking. Co-crystallization means that crystallization of the chemically modified biopolymers (protein and additive) is conducted from solution phase. Alternatively, protein crystals are first grown and then they are chemically modified *via* diffusion of the additives that are soaked into the solvent channels. Both these preparative procedures can be supported by the application of Raman monitoring, particularly by difference Raman spectra (in co-crystallization) or by a time-resolved Raman microscopy

Raman spectroscopy is a vibrational spectroscopy based on the anelastic scattering of a monochromatic light (frequency ν) due to the interaction with the sample. The polarizability tensor of the sample oscillating with a normal frequency ν0, that is associated to the *omni* present molecular vibrations, interacts with the electric field of the laser light. This produces an induced electric dipole oscillating (then irradiating) with three distinct frequency (ν, ν+ν<sup>0</sup> and ν- ν0). The strong elastic scattering at frequency ν0 is the Rayleigh scattering that usually is cut in a Raman experiment. The two minor inelastic components are Raman Stokes bands (ν-ν0) and Raman antiStokes (ν+ν0). At room temperature only Raman stokes bands are intense enough to be recorded. The difference between the wavenumber of the incident light and the scattered Raman band is the *Raman shift* (the x-axis of any Raman spectrum). The yaxis is usually reported in arbitrary units, so that accurate quantitative analysis can be based only on ratio of two distinct bands or by using some internal standard. In the Stokes Raman region (at wavelength lower than the excitation line) also fluorescence is collected. Therefore the counts read in the y-axis is always the sum of Raman and fluorescence, that should be

We should point out that two major kinds of Raman spectra can be collected, depending on the laser line (off-resonant Raman and resonant Raman spectra). In off-resonance Raman spectra no relation exists between excitation line and electronic absorption condition. On the contrary, resonance Raman spectra are collected when a particular excitation wavelength is used, namely within one electronic absorption band of the sample. Resonance Raman spectra are much more intense (depending on the extinction coefficients of the electronic transition) and selective (only normal mode that couples with the vibronic transition can be

(for soaking procedure).

kept as low as possible.

**2.1 Raman effect** 

**2. Principles of Raman spectroscopy** 

**2.2 Off-resonance and resonance Raman** 

Confocal apparatus allows to get rid of any influence of the cover slip (as in vapour diffusion) or container wells (as in FID or batch crystallization supports). Measurement into drops (both hanging and sitting drop) is particularly feasible. In order to avoid significant scattering from mother liquor, thus reduction in spectral quality, it is better to keep the drop as small as possible, and to use a minimal depth into the crystal (particularly for resonance Raman spectra). Water is a weak Raman scatterer, with a small contribution at 1640 cm−1. Precipitating agents, especially when at high content (PEG, MPD, alcohol) may interfere or not, depending on the spectral region of interest. Agarose and silica gel matrix do not interfere significantly, so crystals grown in gel medium can be used as well.

Despite we deal with solid state we will not consider frequencies of the lattice vibrational modes, that for protein crystals are very low (below the Rayleigh cut). For lysozyme crystals it is 25 cm-1.

We will focus on two distinct experimental setups, namely for *in-situ* and *ex-situ* Raman spectroscopy, referred to simultaneous or not to X-ray diffraction experiments (also reported as on-line and off-line (McGeehan et al, 2011)). Below we present separately these two setups and applications.

#### **3.2 Ex situ Raman experiments**

Most of the analysis to monitor chemical modification of protein crystals can be performed by *ex situ* Raman experiments, which involve distinct acquisition of Raman and X-ray diffraction data. The experiments are, indeed, carried out on a Raman microscope that is physically separated from the X-ray diffractometer. This kind of experiments can be easily performed on a commercial or home-built Raman microscope, and it aims to the definition of the experimental conditions (eg soaking time and reactant/protein molar ratio) for the preparation of derivative protein crystals. Raman spectra on very small drops can be recorded also at low temperatures by using a dedicated autostage (Linkham Co), though care must be taken during cooling to ensure high transparency of the drop and to avoid crystal movement within the drop. When flash freezing is adopted, these impediments are overcome. Crystals monitored at room temperature *via* ex situ apparatus can even be frozen and taken to the X-ray diffractometer for the data collection.

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 397

dedicates one of their beamlines to *in situ* Raman-assisted X-ray biocrystallography (see

A recent review on the microRaman apparatus available at synchrotron facility is reported in (McGeehan et al., 2011). For *in situ* setup we intend that the X-ray diffractometer is integrated with a laser (guided by an optical fiber) that is backscattered to a spectrograph. To this end, crystals are mounted on an usual crystallographic head kept into a cryo-loop under a nitrogen flux (typically at 100 K). After the crystal is centered for X-ray diffraction data collection experiments, the optimal orientation for the collection of the best Raman spectra has to be determined, typically by manually re-orienting the crystal. Once the crystal orientation that results in the best signal-to-noise ratio is found, Raman spectra are acquired prior and after the X-ray diffraction data collection, at the same crystal orientation. This allow to check if any photo-chemistry is induced by the X-ray beam exposure. For offresonance Raman spectra it is particularly important to keep the drop volume low and to avoid interference from the loop (nylon loops should not intersect the Raman laser beam path (Carpentier et al., 2011)). When Raman spectra are collected as a function of the crystal orientation (Raman crystallography), information can be obtained on the orientation order and the variability of the relative intensity of Raman bands can be estimated (Tsuboi et al., 2007). Indeed, both off-resonance (Kudryavtsev et al., 1998) and resonance spectra (Smulevich & Spiro 1990) are mildly affected by crystal orientation. In this case general physical chemistry rather than structural information can be extracted by Raman

**4. Interpretation of Raman spectra of derivative protein crystals** 

relates to the strength constant *k* of the harmonic oscillator *via*

A Raman spectrum provides a lot of information: energy shift, intensity and polarization of the scattered light and width of the peak. We will mostly focus on the Raman shift and intensity features. Each Raman shift corresponds to a permitted vibrational transition that

> 1 2

π μ

where μ is the reduced mass. Therefore high frequency corresponds to either stretching or bending with a high strength constant or involving light atoms, such as hydrogen. The presence of the reduced mass at the denominator in equation 1 justifies the wide use of isotopic replacement (eg H/D) for band assignment. Raman transition corresponds, most likely, to a fundamental transition (from the vibrational ground state to the first excited state). Indeed, overtone and combination bands in Raman spectroscopy are much more unlikely than in Infrared spectroscopy (FT-IR). Furthermore, compared to FT-IR, Raman spectroscopy has very different selection rules, and usually bands that are strong in the IR absorption, are not allowed in Raman scattering. Indeed, the great advantage of Raman when studying biological samples is the very weak contribution from water to Raman

Once Raman spectrum has been recorded, reduction for the temperature correction can be performed (Pernice et al., in press). Unless strictly necessary, as for a variable fluorescence coming from background, no baseline correction should be performed. The position of the

ν

*k*

<sup>=</sup> (1)

below).

crystallography.

**4.1 Raman spectra analysis** 

spectra, compared to IR spectra.

#### **3.3 Raman microscopy apparatus**

The Raman confocal microscope (Jasco, NRS-3100) currently available for *ex-situ* experiments at the Department of Chemistry of the University of Naples 'Federico II' is depicted in Figure 1. One of the 458, 488 and 514-nm lines of an air-cooled Ar+ laser (Melles Griot, 35 LAP 431-220), or 406, 413 and 647 nm lines of a water-cooled Kr+ laser (Coherent, Innova 320) can be injected into an integrated Olympus confocal microscope and focused to a spot size of approximately 2 μm by a 100x or 20x objective. The laser power at the sample depends on the wavelength ranging from 5 to 100 mW. A holographic notch filter is used to reject the excitation laser line. Raman back-scattering is dispersed through a monochromator (2400 or 1200 grooves/mm grating) and collected by a Peltier-cooled 1024 x 128 pixel CCD photon detector (Andor DU401BVI). Frequency shifts are calibrated by using indene, cyclohexane or CCl4 as standard, depending on the spectral range of interest. An acceptable spectral resolution is usually considered 4 cm-1.

Actually, since Raman shift for off-resonant spectra is independent of the excitation wavelength, in principle also IR or UV excitation could be used, though at least different optics and detector (a different apparatus) is required.

Fig. 1. Scheme of Raman apparatus available at the University of Naples "Ferderico II" (Dept of Chemistry) to collect spectra from protein single crystals. Adapted after (Carey & Dong, 2004).

#### **3.4 In situ Raman experiments**

Whether the crystal is on the diffractometer or away from it (*in situ* or *ex situ*), Raman spectra are not affected by it, but of course only *in-situ* analysis reveals radiation damage effects (that could be even reversible (Adam et al, 2009)).

That is the reason for many efforts in the last decades to make Raman microscopes available at synchrotron beamlines: the European Synchrotron Radiation Facility (ESRF) (Carpentier et al. 2007), National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL) (Stoner-Ma et al., 2011) and Swiss Light source (SLS) (Owen et al. 2010) part-time dedicates one of their beamlines to *in situ* Raman-assisted X-ray biocrystallography (see below).

A recent review on the microRaman apparatus available at synchrotron facility is reported in (McGeehan et al., 2011). For *in situ* setup we intend that the X-ray diffractometer is integrated with a laser (guided by an optical fiber) that is backscattered to a spectrograph. To this end, crystals are mounted on an usual crystallographic head kept into a cryo-loop under a nitrogen flux (typically at 100 K). After the crystal is centered for X-ray diffraction data collection experiments, the optimal orientation for the collection of the best Raman spectra has to be determined, typically by manually re-orienting the crystal. Once the crystal orientation that results in the best signal-to-noise ratio is found, Raman spectra are acquired prior and after the X-ray diffraction data collection, at the same crystal orientation. This allow to check if any photo-chemistry is induced by the X-ray beam exposure. For offresonance Raman spectra it is particularly important to keep the drop volume low and to avoid interference from the loop (nylon loops should not intersect the Raman laser beam path (Carpentier et al., 2011)). When Raman spectra are collected as a function of the crystal orientation (Raman crystallography), information can be obtained on the orientation order and the variability of the relative intensity of Raman bands can be estimated (Tsuboi et al., 2007). Indeed, both off-resonance (Kudryavtsev et al., 1998) and resonance spectra (Smulevich & Spiro 1990) are mildly affected by crystal orientation. In this case general physical chemistry rather than structural information can be extracted by Raman crystallography.

#### **4. Interpretation of Raman spectra of derivative protein crystals**

#### **4.1 Raman spectra analysis**

396 Current Trends in X-Ray Crystallography

The Raman confocal microscope (Jasco, NRS-3100) currently available for *ex-situ* experiments at the Department of Chemistry of the University of Naples 'Federico II' is depicted in Figure 1. One of the 458, 488 and 514-nm lines of an air-cooled Ar+ laser (Melles Griot, 35 LAP 431-220), or 406, 413 and 647 nm lines of a water-cooled Kr+ laser (Coherent, Innova 320) can be injected into an integrated Olympus confocal microscope and focused to a spot size of approximately 2 μm by a 100x or 20x objective. The laser power at the sample depends on the wavelength ranging from 5 to 100 mW. A holographic notch filter is used to reject the excitation laser line. Raman back-scattering is dispersed through a monochromator (2400 or 1200 grooves/mm grating) and collected by a Peltier-cooled 1024 x 128 pixel CCD photon detector (Andor DU401BVI). Frequency shifts are calibrated by using indene, cyclohexane or CCl4 as standard, depending on the spectral range of interest. An acceptable

Actually, since Raman shift for off-resonant spectra is independent of the excitation wavelength, in principle also IR or UV excitation could be used, though at least different

Fig. 1. Scheme of Raman apparatus available at the University of Naples "Ferderico II" (Dept of Chemistry) to collect spectra from protein single crystals. Adapted after (Carey &

Whether the crystal is on the diffractometer or away from it (*in situ* or *ex situ*), Raman spectra are not affected by it, but of course only *in-situ* analysis reveals radiation damage

That is the reason for many efforts in the last decades to make Raman microscopes available at synchrotron beamlines: the European Synchrotron Radiation Facility (ESRF) (Carpentier et al. 2007), National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL) (Stoner-Ma et al., 2011) and Swiss Light source (SLS) (Owen et al. 2010) part-time

**3.3 Raman microscopy apparatus** 

spectral resolution is usually considered 4 cm-1.

Dong, 2004).

**3.4 In situ Raman experiments** 

effects (that could be even reversible (Adam et al, 2009)).

optics and detector (a different apparatus) is required.

A Raman spectrum provides a lot of information: energy shift, intensity and polarization of the scattered light and width of the peak. We will mostly focus on the Raman shift and intensity features. Each Raman shift corresponds to a permitted vibrational transition that relates to the strength constant *k* of the harmonic oscillator *via*

$$\nu = \frac{1}{2\pi} \sqrt{\frac{k}{\mu}} \tag{1}$$

where μ is the reduced mass. Therefore high frequency corresponds to either stretching or bending with a high strength constant or involving light atoms, such as hydrogen. The presence of the reduced mass at the denominator in equation 1 justifies the wide use of isotopic replacement (eg H/D) for band assignment. Raman transition corresponds, most likely, to a fundamental transition (from the vibrational ground state to the first excited state). Indeed, overtone and combination bands in Raman spectroscopy are much more unlikely than in Infrared spectroscopy (FT-IR). Furthermore, compared to FT-IR, Raman spectroscopy has very different selection rules, and usually bands that are strong in the IR absorption, are not allowed in Raman scattering. Indeed, the great advantage of Raman when studying biological samples is the very weak contribution from water to Raman spectra, compared to IR spectra.

Once Raman spectrum has been recorded, reduction for the temperature correction can be performed (Pernice et al., in press). Unless strictly necessary, as for a variable fluorescence coming from background, no baseline correction should be performed. The position of the

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 399

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.

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

(after Vergara et al., 2008).

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 Raman (Carey, 2006).
