**2. Infrared spectroscopy of water**

structure of proteins, nucleotides, carbohydrates and other biopolymers. The solvent orga‐ nization around the solute macromolecule structures allows folding to the native and func‐ tional conformation and the development of many biological functions such as for example, substrate recognition and binding to an enzyme, protein subunits assembling and to origi‐ nate and stabilize higher order structures such as membranes. A lot of biochemical reactions fundamental in metabolism and synthesis are involving water as universal reagent, such as hydrolysis, condensation, reduction and oxidation. Despite the great interest in the problem, the relationship between the biopolymer conformation and the structure of the water net‐ work cannot still be described with confidence. In the case of proteins, it is well‐known that polypeptide molecules are surrounded in the cell by a hydration shell which can be described as formed by differently interacting water molecules organized in layers surrounding macro‐ molecule. The simplified description of the hydration shells formed by a uniform layering of water molecules around the macromolecule is not realistic: most proteins, for example, offer a surface where binding sites, cleft or crevices provide favorable environments for solvent mol‐ ecules. Such water molecules are organized in clusters or patches decorating the macromol‐ ecule surface and are called "bound" water due to the restricted mobility with respect to the water molecules in bulk. Orientation changes may be related to structural or conformational differences among macromolecules. The bond strength variety of bound water affects mobil‐

A variety of experimental techniques were introduced and applied to study the hydration properties of biological macromolecules: DSC (differential scanning calorimetry), NMR (nuclear magnetic resonance), neutron and X‐ray diffraction, gravimetric techniques, as well as UV‐vis and CD spectroscopies have been employed to characterize the extent of water‐ biomolecules interaction. In addition, the problem of water interactions was object of theoreti‐

Fourier transform IR absorption spectroscopy represents a powerful method to gain struc‐ tural information on hydrated biological macromolecules, alternative to other well‐estab‐ lished techniques whose application presents severe limitation to dry compounds or in the first hydration events. It requires a minimal sample amount and preparation, and it can be used in a wide variety of conditions and geometries. It enables to study (1) the amplitude and position changes of the main absorption bands associated with characteristic functional macromolecule groups, as a function of water content; (2) the properties of water structured

O displays a strong IR spectrum with three main bands corresponding to the OH stretch‐ ing, bending and libration modes. Their contribution can be identified in the spectrum of macromolecules and changes in the hydration conditions significantly influence the infra‐ red spectral pattern. The spectral changes observed as a function of water removal may be monitored, correlated to the changes in macromolecule conformation and used to identify the sites of water sorption. The OH stretching band, in particular, by appropriate mathematic manipulation, can be used to build the water adsorption‐desorption isotherms describing the

O molecules as probes to detect conformational changes in

cal analysis and structure prediction as well as molecular dynamics simulations.

the macromolecule structure induced by water interactions [8].

hydration processes governing each water population.

ity, reorientation and vibrational properties [2–7].

192 Fourier Transforms - High-tech Application and Current Trends

around the biomolecules, using H2

H2

Water has a strong absorbance in the infrared [11–13]. The characteristic normal modes of the H2 O molecule are due to vibrating O–H bond whose frequencies are critically dependent on the aggregation state of water: gas, liquid and solid. In the medium infrared wave num‐ ber region (MIR), the isolated water molecule (gas phase) has three main normal modes of vibration. The symmetric and asymmetric OH stretching vibrations (ν<sup>1</sup> , ν<sup>3</sup> ) have band maxi‐ mum at 3656 and 3755 cm−1 and the bending mode, δ(OH), has the band center at 1594 cm−1. Upon formation of hydrogen bonds in the liquid phase and because of the broadening of the spectral features, ν<sup>1</sup> and ν<sup>3</sup> features are collapsing to a diffuse absorption band that appears 200–400 cm−1 shifted towards high wave number region with respect to the gas phase values. In addition, ν(OH) band width at half‐maximum (FWHM) will broaden as a consequence of the H bond pattern formation. In general, the more heterogeneous are the molecular envi‐ ronments of hydrogen‐bonded molecules, the broader the band. For this reason, the liquid water spectrum is broader than that found for ice spectrum reflecting the vibrations of more selected hydrogen bond energies and configurations. The bending mode shifts towards high wave number region as a consequence of formation of hydrogen bonds: liquid water and ice display bending absorption bands at 1645 and 1670 cm−1, respectively. Libration modes νL are other vibrational modes of condensed phase water. They appear at 685 cm−1 for liq‐ uid water and shifts at 830 cm−1 for ice. Moreover, an absorption band is observed around 2200 cm−1, centered at 2100 cm−1 for liquid water and around 2255 cm−1 for different phases of ice. It represents a combination band, due to the association of the bending δ and libration νL features.

### **2.1. OH stretching band (ν(OH))**

The IR OH stretching band has been widely studied to investigate the properties of water structured around biomolecules. The first pioneering work concerning the study of hydration water of globular proteins was performed by Buontempo, Careri and Fasella in 1972 by ana‐ lyzing the differential Infrared band near 3300 cm−1 in the spectrum of Lysozyme and Bovine Serum Albumin before and after dehydration under various conditions [14]. The study evi‐ denced the presence of water molecules differently mutually interacting and with the protein surface, being possible to distinguish a contribution due to liquid water and a component corresponding to the so‐called tightly bound water. In the following years, thanks to the implementation of the interferometric spectrophotometers instead of the dispersive one, mea‐ surements were performed with a much better sensitivity and reliability. The enhancement in the peak fitting procedures with help of second derivative operation and iteration procedures allowed to improve the qualitative picture by fitting the band into component sub‐bands related to different local H bonding structure. In the paper of Onori and Santucci [15] and Mallamace et al. [16], the best fit deconvolution of the band was performed in the study of, respectively, AOT hydrated micelles and lysozyme at different temperatures to investigate structural dynamical transitions.

Pure water ν(OH) band (3800–2800 cm−1) cannot be fitted by a single Gaussian line, but can be deconvoluted into components, usually assigned to different sets of molecules. Following the central limit theorem stating that the distribution of the sum (or average) of a large number of independent, identically distributed variables will be approximately nor‐ mal, regardless of the underlying distribution, the component number could be enormously large. However, the structural constraints settled on the basis of the number and strength of hydrogen bonds in different arrangements avoids effects of overfitting, allowing the break‐ down of the band up to a maximum of six sub‐bands in dependence on the model assumed to describe water system. The structure of *ν*(OH) band is described in details by Schmidt and Miki [17] and directly related to the O‐H bond lengths: variations in the bond lengths are caused by the influence of the surrounding hydrogen‐bonded network of water molecules and affect position and width of the components bands. According to this view, from the higher wave number region towards the lower one, the first peak (3680 cm−1) can be attrib‐ uted to monomer‐like vibration, due to free O–H vibration of H2 O molecules behaving as in the vapor state. The three intermediate components contribute 85% to the total signal, indi‐ cating that the majority of water molecules have a local hydrogen‐bonded network which includes 3–7 H2 O molecules as confirmed by theoretical calculations. Solid clusters (6–10 H2 O) is responsible for the envelope of the two low wave number bands. The peak position of each band was related to O–H bond length. The values were calculated by transforming the frequency and full‐width half‐maximum values of the *ν*(OH) band components using Badger's rule [18].

In recent times, we have developed a technique operating the removal from the spectrum of the features due to the spurious vibrational bands unrelated to water by means of the operation of subtraction of the "dry sample" spectrum, i.e., the spectrum of the sample gently dehydrated to expel as much solvent as possible without affecting the molecule structure [9, 10, 19]. The cleaned band obtained by such procedure can be treated as a water ν(OH) band and therefore compared with the corresponding band of pure water and analyzed by decon‐ volution in component bands, each one related to different hydrogen bond engagements and lengths.
