Spectrum Analysis, Raman

As well as being crucial in providing complementary information in the study of the crystal structures of certain biological macromolecules, the Cryobench can be used in applications of more general use. Heavy-atom soaking can be monitored if it produces a species with a specific spectroscopic signature (e.g. a mercury–sulfur covalent bond), thus allowing a precise determination of optimal (at least in terms of heavy-atom binding) soaking times (Carpentier et al., 2007 ▶ ); the exposure of crystals to UV light can be used to locate crystals in loops by their intrinsic fluorescence (Jacquamet et al., 2004 ▶ ; Vernede et al., 2006 ▶ ) or to deliberately induce radiation damage to crystals, which can then be exploited to provide phase information for crystal structure solution via UV-RIP (Nanao & Ravelli, 2006 ▶ ); the pH in a crystal can be directly determined by soaking samples in an exogenous pH-sensitive fluorophore (Bourgeois et al., 2002 ▶ ; Fioravanti et al., 2003 ▶ ). Under favourable circumstances, optical spectroscopy can also be used to identify unknown ligands that are found in electron-density maps to be bound to a protein. Examples here include the use of Raman spectroscopy to unambiguously prove the binding of a nitrate ion to xylose isomerase (Carpentier et al., 2007 ▶ ) and the identification of chlorophyll a and carotenoids, in substoichiometric amounts, in crystals of the c-ring of a proton-coupled F₁F_o ATP synthase (Pogoryelov et al., 2009 ▶ ).
The Cryobench is also an indispensible tool for kinetic crystallography (KX) experiments based on the use of caged compounds. Synchronization of de-caging can be achieved with an actinic light. In this regard, noncoloured proteins can be made coloured in the near-UV range by chemically grafting a photolabile group onto either a substrate (e.g. deoxy­thymidine monophosphate), cofactor (e.g. adenosine triphos­phate or dioxygen) or product [e.g. (arseno)choline] of the protein (Colletier et al., 2007 ▶ ; Howard-Jones et al., 2009 ▶ ; Specht et al., 2001 ▶ ; Ursby et al., 2002 ▶ ). Less expectedly, the Cryobench can also be used in experiments that use KX to study the catalysis of inorganic complexes. Because crystals of small molecules allow very little movement of the molecules that they contain, KX experiments on such systems are difficult to perform. However, in an elegant approach, reaction-intermediate states of an inorganic iron complex, as monitored using UV–vis absorption and Raman spectroscopies, were trapped using crystals of a protein with a large cavity and their three-dimensional structures were solved (Cavazza et al., 2010 ▶ ).
A final advantage of the Cryobench is that the temperature at which measurements are carried out can be varied. In order to prepare KX experiments, temperature-derivative fluorescence, or absorbance, microspectrophotometry (TDFM/TDAM) has been developed to allow the monitoring of solvent phase transitions in protein crystals (Weik et al., 2004 ▶ ) and, in protein solutions, to determine whether the correlation between solvent and protein motions is necessary for the formation of reaction-intermediate states (Durin et al., 2009 ▶ ).

To each tube containing the pellet of non-deuterated or deuterated bacterial suspension, 200 μL of sterilized MilliQ water was added. One microliter was taken and placed on a clean quartz slide. The slide was then placed in the Raman system. A detailed description can be found in the works of these authors [16 (link)]. Briefly, the system allows targeting of bacterial cells thanks to imaging modalitied, and the measurement of single-cell Raman spectra using a confocal arrangement. The beam of a 532 nm, 50 mW laser (Spectra Physics Excelsior 532-50-CDRH) is attenuated and focused by a microscope objective (×100, 0.8 NA, Olympus LMPLFLN) in order to provide a spot size of 1 μm in diameter at the sample. Raman back-scattered light from an individual bacterium is collected by the same objective, filtered from Rayleigh light by a notch filter (NF03-532E, Semrock, New York, USA), and focused into the entrance fiber of a dispersive spectrometer (Hyperflux U1-532, Tornado Spectral systems, Toronto, Canada). The spectrometer featured at −15°C TE-cooled CCD, and spectral resolution of 10 cm^-1 over the band 500–3400 cm^-1. For each analysis point, namely a given configuration in terms of incubation time and D₂O concentration, at least 60 bacteria cells were targeted and the corresponding Raman spectra were acquired. The Raman spectroscope acquisition parameter for each spectrum was tuned to 25 seconds and 250 mW, with our system that is a Lab custom system, not optimized for later industrial purpose.