Resonance Raman Spectroscopy

Abstract

Resonance Raman spectroscopy is a vibrational spectroscopic technique that utilises electronic absorption bands to dramatically enhance the magnitude of the signal. It has been used effectively to study protein structure and function. Resonance Raman spectroscopy performed in the UV spectral range can probe the structure of proteins by monitoring the local environment of aromatic side chains. Deep‐UV resonance Raman spectroscopy with excitation wavelengths less than 200 nm can be used to determine secondary structure of proteins by monitoring the frequencies and intensities of amide backbone modes. The ability to monitor both proteins backbone and side chains in kinetic investigations makes this a very powerful technique for investigations of protein folding and amyloid formation.

Key Concepts:

  • Resonance Raman spectroscopy measures molecular vibrations, which provides information on local environment and H‐bonding. The resonance effect leads to selective enhancement and a simplified spectrum.

  • Resonance Raman spectroscopy can be used to study samples in a variety of different physical forms such as solutions, gels and fibres.

  • Time‐resolved techniques involving resonance Raman spectroscopy span a wide time range from picoseconds to seconds. Selective investigation of chromophores, protein backbones and sidechains provides a wealth of dynamic information.

  • Secondary structure of proteins can be determined using UV resonance Raman spectroscopy, which monitors the amide vibrational modes of the peptide backbone. In particular the amide III3 mode frequency has a sinusoidal dependence on the Ramachandran Ψ angle.

  • UVRR spectroscopy is increasingly being used to study protein folding and the association of proteins and peptides into amyloid fibres.

Keywords: vibrational spectroscopy; proteins; time‐resolved; secondary structure conformation; temperature jump

Figure 1.

Schematic illustration of the differences between the IR, Raman and resonance Raman spectroscopic methods. As shown, all the three methods are measuring the 0→1 vibrational transition in the ground electronic state. In the case of the resonance Raman spectroscopy, the use of an excitation wavelength that is the same frequency as an electronic absorption transition leads to strong enhancement of the signal.

Figure 2.

Example of the enhancement effect and selectivity of the resonance Raman spectroscopy. For the protein haemoglobin, excitation into the absorption band at 420 nm yields a vibrational spectrum that mainly contains contributions from the haem moieties. Excitation into the absorption band at 230 nm selectively enhances the vibrational contribution from the Tyr and Trp amino acid residues in the protein.

Figure 3.

Schematic illustration of a potential instrument layout for the time‐resolved RR spectroscopy. As shown, the timing of the pulses from the pump and probe laser are controlled electronically and are synchronised with a computer or pulse generator. The use of a dichroic mirror allows the two beams to travel coincidentally to the sample. The pump beam initiates the reaction and the probe beam is used to obtain an RR spectrum of the sample at a set delay time after the pump. For temperature‐jump methods, the pump beam is typically in the IR range.

Figure 4.

Example of how the UVRR spectroscopy can be used to monitor fibril formation. Two‐dimensional correlation spectroscopy is used to show that for lysozyme random coil formation is asynchronously correlated with β‐sheet formation. The negative peak associated with the β‐sheet frequencies indicates that the β‐sheet forms after random coil. Reprinted with permission from Shashilov et al.. Copyright 2007 American Chemical Society.

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Further Reading

Austin JC, Jordan T and Spiro TG (1993) Ultraviolet resonance Raman studies of proteins and related model compounds. In: Clark RJH and Hester RE (eds) Biomolecular Spectroscopy, pp. 55–127. New York: John Wiley and Sons Ltd.

Ferraro JR, Nakamoto K and Brown CW (2003) Introductory Raman Spectroscopy. Boston: Academic Press.

Kincaid JR (1995) Structure and dynamics of transient species using time‐resolved resonance Raman spectroscopy. Methods in Enzymology 246: 460–501.

Rousseau DL and Han S (2002) Time‐resolved resonance Raman spectroscopy of intermediates in cytochrome oxidase. Methods in Enzymology 354: 351–368.

Takeuchi H (2011) UV Raman markers for structural analysis of aromatic side chains in proteins. Analytical Sciences 27(11): 1077–1086.

Varotsis C and Babcock GT (1993) nanosecond time‐resolved resonance Raman spectroscopy. Methods in Enzymology 226: 409–431.

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How to Cite close
Mukerji, Ishita(Dec 2012) Resonance Raman Spectroscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003113.pub2]