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

Antonello Merlino, Filomena Sica and Alessandro Vergara\* *Department of Chemistry, University of Naples "Federico II", Naples, Italy Istituto di Biostrutture e Bioimmagini, CNR, Naples Italy* 

## **1. Introduction**

392 Current Trends in X-Ray Crystallography

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> Below to crystallographic applications, protein crystals are of great interest in other numerous fields of biology and biotechnology. Cataract, or the loss of transparency of the eye lens, is related to the alteration of physical, chemical, and structural properties of proteins of the crystallin family, that may lead to crystallization under some physiological conditions (Tardieu, 1998). Other pathological states are known to be a consequence of the *in vivo* formation of crystals, made of either proteins or other macromolecular assemblies. Examples are viral proteins stored in plant cells, viral particles in animal cells, hemoglobin C and S causing anemia, or ribosomal particles accumulating in the brain of patients suffering from *presenile dementia* (McPherson, 1999).

> Of course, crystals of biological macromolecules that are prepared *in vitro* have important applications: they are tools to obtain atomic models of the molecules and to design specific ligands and new drug formulations. Medicinal formulations composed of either insulin (Richards et al., 1999) or α-interferon crystals (Reichert et al., 1996) are already applied in treatments to ensure the continuous release of protein in blood. Crystallographic analysis of highly ordered crystals with intense X-ray sources provides accurate three dimensional structures (Ducruix & Giegé, 1999). The success of this technique strictly depends on obtaining diffraction-quality crystals. The process of crystallization remains a hit-and-miss affair, typically involving screening hundreds of conditions. The crystallization of biological macromolecules shares many common properties with those of small solute molecules (e.g. growth by 2D nucleation or by screw dislocation mechanisms), but their crystals exhibit several peculiarities: most of them have a high solvent content (e.g. 30-80 vol%), few intermolecular contacts, and a high density of defects (Malkin et al., 1996).

> Briefly, protein crystallization requires the formation of a supersaturated protein-precipitant solution. The transition from a stable solution to a supersaturated one can be achieved by increasing the concentration of precipitant and/or that of protein (Vergara et al., 2003). The most frequently used crystallization method is the *vapor diffusion technique*. A drop containing protein, buffer, salt and precipitant is equilibrated against a reservoir (buffer, salt and precipitant). The difference in concentration between the drop (lower) and the reservoir (higher) drives the system toward equilibrium by diffusion through the vapor phase. The drop can either be placed on the underside of the cover slide (*hanging drop*) or placed on a

Monitoring Preparation of Derivative Protein Crystals *via* Raman Microscopy 395

enhanced) than off-resonance Raman spectra. A more extensive presentation of the quantum

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

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

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

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

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

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

interfere significantly, so crystals grown in gel medium can be used as well.

and taken to the X-ray diffractometer for the data collection.

mechanics of the Raman effect is elsewhere reported (Long, 2002).

straight analysis on the sample.

**3.1 Crystal sampling** 

it is 25 cm-1.

setups and applications.

**3.2 Ex situ Raman experiments** 

measurements requires standard handling.

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

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 (for soaking procedure).

#### **2. Principles of Raman spectroscopy**

#### **2.1 Raman effect**

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 kept as low as possible.

#### **2.2 Off-resonance and resonance Raman**

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 enhanced) than off-resonance Raman spectra. A more extensive presentation of the quantum mechanics of the Raman effect is elsewhere reported (Long, 2002).
