FTIR / Raman spectroscopy

Fundamentals of vibration spectroscopy

To understand the basics of vibrational spectroscopy, each molecule can be thought of as a collection of several masses (the atoms) and connecting elastic springs (the atomic bonds). Along each spring the connected masses can oscillate. The respective oscillations depend on the spring, the masses involved and the angle between the springs, and are highly characteristic. The resulting oscillation spectra can be measured in the infrared spectral range in absorption and with Raman spectroscopy in scattering.

In contrast to this classical model (mass spring), the quantum mechanical model excites discrete vibration levels. Therefore, one speaks of vibration spectra. The figure above shows the stretching and bending vibration of the water molecule and the two resulting IR absorption bands. These two bands are characteristic for a water molecule. The resulting spectra are therefore characteristic for the molecule in question and comparable to fingerprints, which can be used to identify the molecules label-free.

Molecular resolution in proteins

With small molecules, the individual bands can be clearly assigned to the respective oscillations of the molecule. In proteins, FTIR (Fourier Transform Infrared) difference spectroscopy can be used to select the bands of the active centre from the background absorption of the total protein (129). Static FTIR difference spectroscopy was introduced by Siebert, Mäntele, and Gerwert in 1983 (2). This involves freezing an intermediate at low temperatures. The individual difference bands could for the first time be clearly assigned to specific molecular groups, such as aspartic acids using isotope labelling (3,8), or even more precisely, to a single aspartic acid using site-specific mutagenesis (19).

This was the decisive breakthrough of the method, which was initially viewed very critically because only very small changes are selected against the strong background absorption of the entire protein, similar to a needle in a haystack. An important next milestone was the establishment of time-resolved FTIR differential spectroscopy, which allows the simultaneous measurement of the different reactions in a protein at room temperature in a time-resolved manner and thus the protein dynamics (13, 14, 18, 20). Based on time-resolved FTIR spectroscopy, it is now possible to produce films of protein dynamics complementary to X-ray structure analysis, which provides static images of the ground state. The precise simultaneous start of the protein reactions is crucial for difference spectroscopy. This method is therefore particularly suitable for proteins with chromophores, because in these cases the reaction can be started with a laser flash (105, 158). By introducing photolabile, so-called “caged” compounds, such as caged GTP, the method could be extended to non-chromophoric proteins (59, 169).