How to study proteins by circular dichroism

doi:10.1016/j.bbapap.2005.06.005

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Volume 1751, Issue 2, 10 August 2005, Pages 119-139

https://doi.org/10.1016/j.bbapap.2005.06.005 Get rights and content

Abstract

Circular dichroism (CD) is being increasingly recognised as a valuable technique for examining the structure of proteins in solution. However, the value of many studies using CD is compromised either by inappropriate experimental design or by lack of attention to key aspects of instrument calibration or sample characterisation. In this article, we summarise the basis of the CD approach and its application to the study of proteins, and then present clear guidelines on how reliable data can be obtained and analysed.

Introduction

Since the late 1980s, there has been an explosive growth in structural biology with the number of high resolution structures of proteins added to the protein data bank (PDB) currently growing at more than 2000 per year. This has allowed much more detailed insights into the function of systems of ever-increasing size, including complex cellular assemblies such as the proteasome [1] and the large ribosomal subunit [2]. To a large extent, the growth in structural biology has been driven by developments in recombinant DNA technology which allow proteins to be produced in the (often substantial) quantities required, as well as by advances in data analysis and bioinformatics. However, there is a growing realisation of the need to perform structural studies under the conditions in which proteins actually operate (i.e., generally in solution), as well as under other conditions and to provide measures of the rates of structural changes of proteins, which are often essential to their biological function. Circular dichroism (CD) has become increasingly recognised as a valuable structural technique for addressing these issues. A significant improvement in the provision of CD instrumentation has occurred in recent years; unfortunately, it is not always clear that such instruments are being used to their best advantage. The aim of this article is to provide a brief summary of the CD technique and its applications with particular reference to the study of proteins. It will then go on to address the important practical aspects of performing CD experiments on proteins and provide clear guidance as to how reliable data can be obtained and interpreted. We hope that the article will help users to avoid most of the common errors which, regrettably, occur all too frequently in the published literature. This article is confined to the CD of electronic transitions in molecules (electronic CD, ECD); for details of more specialist aspects of CD such as vibrational CD (VCD) or fluorescence-detection CD (FDCD) other articles should be consulted [3], [4].

Section snippets

Origin of the CD effect

Plane polarised light can be viewed as being made up of 2 circularly polarised components of equal magnitude, one rotating counter-clockwise (left handed, L) and the other clockwise (right handed, R). Circular dichroism (CD) refers to the differential absorption of these 2 components (see Fig. 1). If, after passage through the sample being examined, the L and R components are not absorbed or are absorbed to equal extents, the recombination of L and R would regenerate radiation polarised in the

Information available from CD studies of proteins

CD signals only arise where absorption of radiation occurs, and thus spectral bands are easily assigned to distinct structural features of a molecule. An advantage of the CD technique in studies of proteins is that complementary structural information can be obtained from a number of spectral regions.

In proteins, the chromophores of interest include the peptide bond (absorption below 240 nm), aromatic amino acid side chains (absorption in the range 260 to 320 nm) and disulphide bonds (weak

Preparation

Currently, the majority of protein samples are produced by over-expression of the gene encoding the protein in a suitable host system [42], [43]. Such systems are usually bacteria such as Escherichia coli, lower eukaryotes such as yeast, or insect cells such as the fall army-worm Spodoptera frugiperda. The choice of host system is dictated by a number of factors including the size of the polypeptide chain (large chains often form insoluble inclusion bodies in bacteria) and the need to ensure

Units of CD data

CD data are presented in terms of either ellipticity [θ] (degrees) or differential absorbance (ΔA). The data are normalised by scaling to molar concentrations of either the whole molecule or the repeating unit of a polymer.

For far UV CD of proteins, the repeating unit is the peptide bond. The Mean Residue Weight (MRW) for the peptide bond is calculated from MRW = M/(N − 1), where M is the molecular mass of the polypeptide chain (in Da), and N is the number of amino acids in the chain; the number of

Determination of protein concentration

As mentioned in Section 3.1, accurate determination of protein concentration is particularly important for reliable determination of secondary structure by far UV CD.

Wavelength calibration of spectropolarimeter

A number of methods are available for calibrating the wavelength of the CD instrument, including the use of rare earth element filters (e.g., holmium oxide which has peaks at 279.4 nm, 361.0 nm and 453.7 nm), benzene vapour (241.7 nm, 253.0 nm and 260.1 nm) and neodymium glass (586.0 nm). In addition solutions used for calibrating the magnitude of the CD signal (Section 7.2) such as CSA or pantolactone can be used to check wavelength calibration. Miles et al. [68] recommend that an instrument

Instrument parameters

Various machine settings can be adjusted to improve the results

•
bandwidth;
•
time constant;
•
scan rate;
•
number of scans.

The following considerations are important in selecting these parameters:

Spectropolarimeter

The spectropolarimeter should be sited in a room where the temperature and humidity are kept reasonably constant (this may involve air-conditioning) and which is not subject to excessive mechanical vibration or the accumulation of atmospheric dust.

The high intensity light source will convert residual O₂ to O₃ which damages the optics. It is recommended that the instrument should be purged with N₂ at about 10–15 l/min for 15 min before switching on the light source.

Cylinders of O₂-free N₂ are

Assessment of the reliability of secondary structure analysis by CD

When assessing the secondary structure estimates for a given protein, it is important to bear in mind that there are several different algorithms available (Section 3.1) which use different computational approaches and may employ distinct data sets of reference proteins obtained over various spectral ranges. This makes it difficult to give hard and fast rules which apply under all circumstances. However, the reliability of the analysis can be judged by keeping the following points in mind [10],

Summary of key points for reliable data collection and analysis

•
Make sure that the protein is characterised; check its identity and purity (Section 4.1.2). Determine the concentration of the protein solution accurately (Section 6).
•
Make sure the protein is dissolved in a suitable solvent system in which it is stable and which, ideally, makes only small contributions to the overall absorbance of the sample over the wavelength range of interest (4.2 Choice of solvent/buffer system for CD studies, 8.2 Choice of correct protein concentration and cell pathlength

Acknowledgements

The work of the CD facility at Glasgow has been supported by the Biotechnology and Biological Sciences Research Council of the U.K. We would like to thank Bonnie Wallace, Alison Rodger and Liz Rideout for helpful discussions and providing unpublished data, and Robert Matthews and Jill Zitzewitz for providing data files.

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ReviewHow to study proteins by circular dichroism

Abstract

Introduction

Section snippets

Origin of the CD effect

Information available from CD studies of proteins

Preparation

Units of CD data

Determination of protein concentration

Wavelength calibration of spectropolarimeter

Instrument parameters

Spectropolarimeter

Assessment of the reliability of secondary structure analysis by CD

Summary of key points for reliable data collection and analysis

Acknowledgements

Anal. Biochem.

Methods Enzymol.

Anal. Biochem.

Anal. Biochem.

Curr. Opin. Chem. Biol.

J. Mol. Biol.

Anal. Biochem.

Adv. Prot. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Methods Enzymol.

Neurodegeneration

Trends Biochem. Sci.

Methods

J. Mol. Biol.

Anal. Biochem.

Methods

J. Biol. Chem.

Biochim. Biophys. Acta

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

Structure of 20S proteasome from yeast at 2.4 Å resolution

Nature

The structural basis of ribosome activity in peptide bond synthesis

Science

Circular Dichroism and Linear Dichroism

The use of circular dichroism in the investigation of protein structure and function

Curr. Prot. Pept. Sci.

On the analysis of membrane protein circular dichroism spectra

Protein Sci.

Analyzing protein circular dichroism spectra for accurate secondary structures

Proteins: Struct. Funct. Genet.

Evaluation of secondary structure of proteins from UV circular dichroism using an unsupervised neural network

Protein Eng.

Estimation of globular protein secondary structure from circular dichroism

Biochemistry

DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra

Bioinformatics

DICHROWEB, An online server for protein secondary structure analyses from circular dichroism spectroscopic data

Nucleic Acids Res.

Analyzing protein circular dichroism spectra for accurate secondary structures

Proteins: Struct. Funct. Genet.

Review
How to study proteins by circular dichroism