Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

Abstract

Principally through the efforts of crystallographers, we are being presented with an ever expanding atomic view of the biological world. Although this brings into focus many questions regarding the mysteries of function, techniques are needed that facilitate the transition in our understanding from structure to function. Raman spectroscopy is one of these; because the Raman effect involves an intimate interplay between atomic positions, electron distribution, and intermolecular forces, it sits at the bridgehead between structure and function. Thus, the Raman technique can answer questions that lie at the heart of issues such as ligand macromolecule recognition and enzymatic catalysis. Raman spectroscopy involves analyzing the scattered photons from a laser beam focused into the sample solution (1). The inelastic scattered photons (the Raman spectrum) provide information on molecular vibrations that, in turn, yield data on molecular conformation and environment. At its most effective, Raman spectroscopy can provide exquisite detail from an important site in a much larger macromolecular complex. Although Raman was first applied to the definition of biological molecules in the 1930s (2), the giant has remained drowsy due the difficulties both in obtaining high quality data and in interpreting those data. Considerable advances have been made in these areas in the past few years, and the giant is stirring! A major goal of this review is to provide biochemists with enough information to determine whether the Raman technique could provide structural insights into their systems. The specific issues addressed are which type of systems are amenable to study and what information could be obtained. Practically, present-day sample requirements are for 20 l of clear solution, where the target molecule is in the 100 -300 M range. Because the number of vibrational modes of a molecule is 3n Ϫ 6, where n is the number of atoms, the complete Raman spectrum of a macromolecule is exceedingly complex. Thus, Raman is most suited to systems where it is possible to focus upon a small region of interest, e.g. a ligandreceptor or enzyme-substrate binding site. Historically, this condition was achieved by using resonance Raman spectroscopy (1) to obtain the intensity-enhanced spectra from chromophores at specific sites in macromolecules. Recent technical advances mean that similar information can now be gleaned from non-chromophoric systems, markedly broadening the application of the technique. The information obtained can be very detailed, exceeding the level of resolution found in x-ray or NMR analyses (3-5). In addition to providing structural data, the Raman spectrum can also reveal changes in the distribution of electrons in a bound ligand and details of active site-ligand interactions, such as hydrogen bonding strengths.Raman spectroscopy is beginning to fulfill its potential to contribute to structural biology because the three roadblocks that impeded its application to biological systems have been all but removed. These were low se...

Reference database of Raman spectra of biological molecules

“…Over the past few years, its possibilities in biology and microbiology have been explored. As mentioned by Carey,4 improvements have been made concerning problems such as low sensitivity, fluorescence rejection and data treatment, and thus the application of Raman spectroscopy became possible in these research fields.…”

Single-Cell Microbiology: Tools, Technologies, and Applications

“…Raman spectra provide information on molecular vibrational states, which are dependent on the nature of chemical bonding within a molecule or sample (178,261). These spectra yield clues to the types and lengths of chemical bonds present and on the molecular conformation or environment (48,205). Microspectroscopic Raman probes capable of illuminating an area as small as 1 by 1 m enable the characterization of individual cells and their subcellular components (135).…”

“…The Raman effect is an induced emission of light resulting from the inelastic scattering of a small number of photons from a monochromatic light source (48,205,261). Raman spectra provide information on molecular vibrational states, which are dependent on the nature of chemical bonding within a molecule or sample (178,261).…”

Traditional Biomolecular Structure Determination by NMR Spectroscopy Allows for Major Errors

“…Raman spectroscopy has the advantage of not requiring coloured functional groups, and can provide detailed information (Carey, 1999;Hildebrandt & Lecomte, 2000;Tuma, 2005). Raman spectroscopy is analogous to infrared absorption spectroscopy in that it probes the vibrational energy levels of molecules rather than the electronic transitions probed by UV-Vis absorption.…”