To achieve such measurements, conditions must be used so that the total absorbance of the sample (due to protein, buffer and other added components) is below ∼1.2 at all wavelengths. 8,9 To accurately determine the secondary structure of a protein based on CD data, the data obtained must include a spectral range covering, at least, the wavelengths between 240 and 190 nm more accurate results are obtained if data collected includes even lower wavelengths, because more electronic transitions (peaks) will be included, increasing the information content of the data. To optimise the amount of high quality and reproducible CD data obtained from a given sample, it is essential to follow good practice protocols for data collection (see Table 1 for example). This tutorial review discusses not only methods and software currently available for obtaining, processing, validating, and interpreting high quality CD data obtained using lab-based (commercial) CD instruments (as well as synchrotron radiation circular dichroism (SRCD) beamlines) on a variety of samples types, but also the bioinformatics tools and resources available for determining novel details of the structure and function of proteins based on such data. In recent years circular dichroism beamlines have been developed at synchrotron light sources, taking advantage of both their high light flux, which enables faster collection of data from smaller amounts of protein, and the higher information content available due to the lower wavelengths that can be achieved at these high-intensity light sources. This has resulted in its wide-spread use both by the biochemical and structural biology communities to complement the information derived by those other biophysical methods, 4 as well as by the pharmaceutical industry, 6,7 to assess whether a protein is correctly folded, to monitor structural changes induced by interactions with ligands including other proteins, and to determine protein stability under environmental stresses induced by, for example, changes in pH or temperature. Indeed, in the same 10 year time period, nearly half of the publications which included CD spectroscopy also included information derived from at least one of these other techniques.ĬD spectroscopy has a number of advantages with respect to the higher resolution structural techniques such as crystallography, electron microscopy, and NMR spectroscopy in that it requires relatively small amounts of sample under conditions (temperature, concentration and components present) that may be more comparable to those found in cells. The information derived by CD spectroscopy, which can also include information on dynamic changes in solution or in environments such as membranes or films, is often complementary to that produced by other biophysical, computational and chemical methods such as crystallography, cryo-electron microscopy, NMR spectroscopy, FTIR spectroscopy, vibrational circular dichroism (VCD) spectroscopy, 5 molecular dynamics simulations, and low angle scattering. 1–4 In the past decade, more than 24 000 papers have been published using CD spectroscopy to characterise the structures of polypeptides and proteins. mutations), or intermolecular interactions. It has been widely used to discern the secondary structure of proteins based on electronic transitions in the far ultraviolet (UV) wavelength region (∼240 to 170 nm) and to monitor the local tertiary structure environment of aromatic amino acid residues in the near UV region (∼300 to 260 nm), as a function of their physical or chemical environment, amino acid composition ( i.e. Circular dichroism (CD) spectroscopy is an optical spectroscopic method which exploits the differential absorption of left- and right-circularly polarised light by such chromophores, and can be harnessed to derive structural information about protein conformations. 1. Introduction to circular dichroism spectroscopy of proteinsīiomolecules such as proteins are built up of chiral subunits that produce signals when illuminated by circularly-polarised light in the near and far ultraviolet wavelength ranges where the amide and carbonyl groups of the polypeptide backbones absorb.
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