Fingerprinting haemprotein crystal structures

A group of scientists from Europe and the USA have successfully used high-resolution X-ray crystallography alongside resonance Raman Spectroscopy to “fingerprint” and validate different redox and ligand states in crystal structures of proteins in order to gain maximum functional information and to avoid, for example, the misinterpretation of reaction mechanisms. This breakthrough may prevent future inaccurate predictions in the structure and role of proteins. [Kekilli et al. (2014), Acta Cryst. D70, 1289-1296; doi: doi:10.1107/S1399004714004039]

The combined resonance Raman (RR) and crystallographic data described in the paper demonstrate that online RR spectroscopy is a powerful tool for fingerprinting both redox and ligand states in single crystals of haemproteins.This approach allows information on vibrational states to be gained and, if performed on-axis, is particularly useful for large crystals, where UV-visible absorption micro-spectrophotometry is not feasible owing to the opacity of the haemprotein crystals.

Single-crystal spectroscopies particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. This complementary combined approach to structure determination has proved to be a powerful tool to obtain useful data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.

In this study the scientists successfully present a comprehensive, correlated single-crystal resonance Raman and structural study of Alcaligenes xylosoxidans cytochrome c prime (AxCYTcp) in its ferric, ferrous and gas ligand-bound forms as well as characterizing X-ray induced changes to these states. The researchers went on to demonstrate that redox and ligand states in crystal structures can quite effectively be spectroscopically fingerprinted in situ on a macromolecular crystallography beamline, in this case at Swiss Light Source beamline X10SA.

This methodology, if applied routinely to structural studies of haemproteins, has the potential to radically increase and complement the biological significance of the results gained from crystallographic experiments, such as redox-dependent biological mechanisms.

Lead researcher Dr Mike Hough at the School of Biological Sciences, University of Essex, commented, "The resonance Raman technique is applicable to many protein crystals that contain chromophores. UV-visible spectroscopy has been used quite successfully for similar purposes however as a surface technique, resonance is very useful for larger crystals which are often opaque to light and also gives information on vibrational properties".

The technique is robust and could be rolled-out across many X-ray crystallography beamlines or more importantly as an integral part of future beamlines.