Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics

Abstract

The technique of Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy and its advantages over conventional circular dichroism spectroscopy are described in this tutorial review, as well as recent applications of the technique in structural and functional genomics. Circular dichroism (CD) spectroscopy is a well-established method in biological chemistry and structural biology, but its utility can be limited by the low flux of the light source in the far ultraviolet and vacuum ultraviolet wavelength regions in conventional CD instruments. The development of synchrotron radiation circular dichroism (SRCD), using the intense light of a synchrotron beam, has greatly expanded the utility of the method, especially as a tool for both structural and functional genomics. These applications take advantage of the enhanced features of SRCD relative to conventional CD: the ability to measure lower wavelength data containing more electronic transitions and hence more structural information, the higher signal-to-noise hence requiring smaller samples, the higher intensity enabling measurements in absorbing buffers and in the presence of lipids and detergents, and the ability to do faster measurements enabling high throughput and time-resolved spectroscopy.This article discusses recent developments in SRCD instrumentation, software, sample preparation and methods of analyses, with particular emphasis on their applications to the study of proteins. These advances have led to new applications in structural genomics (SG), including the potential for fold recognition as a means of target selection and the examination of membrane proteins, a class of proteins usually excluded from SG programmes. Other SG uses include detection of macromolecular interactions as a screen for complex formation, and examination of glycoproteins and sugar components. In functional genomics (FG) new applications include screening for ligand binding as a means of identifying function, and examination of structural differences in mutant proteins as a means of gaining insight into function.

The role of the N-terminal loop in the function of the colicin E7 nuclease domain

“…the secondary structure and its changes are responsible for this effect in the wavelength region of the UV light (180-250 nm). Since the synchrotron radiation circular dichroism (SRCD) spectroscopy provides an optimal and even flux of the UV light in a highly controlled manner it can be applied for accurate study of the solution structure and interactions of proteins [60].…”

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

“…There are numerous reviews on the theory and use of circular dichroism (CD) [1][2][3][4][5][6][7] as well as a website with animated graphics http://www.enzim.hu/~szia/cddemo/, which illustrates the production of circularly polarized light, the circular dichroism of optically active molecules, and how passing plane polarized light through an asymmetric molecule results in the production of elliptically polarized light. An introduction to CD and a description of how to estimate the secondary structure of a protein from its CD spectrum are in the first protocol in this series 8 .…”

Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly

“…The aim of the present study was to determine if the CTD of NaChBac influences expression of this channel and its assembly into the cell membrane fraction and to elucidate the nature of the CTD. To achieve the latter, we utilized the emerging technique of synchrotron radiation circular dichroism (SRCD) spectroscopy (18), which provides the sensitivity and accuracy needed to examine the structures of a series of closely related truncated constructs of the channel. As a result, this study has also exemplified a new range of possible applications of SRCD spectroscopy in structural biology.…”