Calibration and standardisation of synchrotron radiation and conventional circular dichroism spectrometers. Part 2: Factors affecting magnitude and wavelength

Andrew J. Miles,Jonathan G. Lees,B.A. Wallace,Frank Wien

doi:10.1155/2005/263649

Abstract

Circular dichroism (CD) spectroscopy is an important tool in structural biology, especially for protein secondary structure analyses. Synchrotron radiation circular dichroism (SRCD) spectroscopy is a modified version of the technique that uses the intense light from a synchrotron source to enable the collection of data to much lower wavelengths than possible on conventional circular dichroism (cCD) instruments. There is a need for standardization of calibration methods amongst and between cCD and SRCD instruments to ensure consistency and the ability to use common reference databases for empirical secondary structural analyses. In a previous study (Spectroscopy17(2003), 653–661), we compared optical rotation measurements on several cCD and SRCD instruments, whilst holding constant other experimental factors. In this study, other experimental parameters which contribute to the spectral magnitude, such as cell pathlength and protein concentration determinations, are examined. In addition, the extent of wavelength calibration variations between instruments and their effects on secondary structure calculations have been examined. Hence, this paper provides additional practical guidance for “good practice” in the measurement of CD data.

Highlights

Circular dichroism (CD) spectroscopy is a long-established technique for examining the secondary structure of proteins and for monitoring protein interactions and folding pathways
The intense light from the synchrotron source permits the collection of higher quality data than that obtained using conventional circular dichroism instruments and appreciably extends the lower wavelength limit of the data [3]
Whilst the pathlengths of long cells (0.1–1.0 cm) are usually accurately reported by the manufacturers, the reported pathlengths of short cells can be in error by as much as 50%, which in turn will result in this level of error in the spectral magnitudes

Summary

Introduction

Circular dichroism (CD) spectroscopy is a long-established technique for examining the secondary structure of proteins and for monitoring protein interactions and folding pathways. It is gaining acceptance as a technique approved for the characterization of macromolecules for pharmacological use. It has been suggested that the extra information available in the vacuum ultraviolet (VUV) data will allow more accurate discernment of structural features, potentially even including information on folds and motifs [4] To this end, new reference databases of circular dichroism spectra which include the lower wavelength data and a wider base of protein structural types, are being created

Methods

Results

Conclusion