Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Secondary structure
Optical methods for studying secondary structure

The most reliable way to determine Secondary Structure is through X-ray analysis of a protein's three-dimensional structure. However, this method is complex and time-consuming, lagging significantly behind modern techniques for establishing Primary Structure. Therefore, It is important to at least roughly estimate the proportion of various secondary structure types in a protein. Furthermore, X-ray crystallography does not allow for monitoring Changes in the spatial—and consequently secondary—structure of a protein in solution under conditions particularly relevant to The Study of its function.

Optical Methods, such as circular dichroism (CD) or optical rotatory dispersion (ORD) studies, are employed for this type of task. Both effects rely on electronic interactions between chromophores—the carbonyl groups of adjacent peptide bonds. These interactions depend heavily on the relative orientation of the carbonyl groups within the three-dimensional structure and, therefore, on the conformation of the corresponding segments of the protein's polypeptide chain.

Thus, a specific set of φ and ψ angles characteristic of a particular type of Protein secondary structure corresponds to a distinct circular dichroism spectrum. In the observed spectra of real Proteins, the contributions of individual secondary structure types are additive. Comparing the circular dichroism spectra of a series of proteins that, according to X-ray crystallography data, have significantly different contents of α-helices, β-sheet elements, β-turns, and unordered regions makes it possible to derive the basis spectra characteristic of these secondary structure types. The circular dichroism spectrum of the protein under study can then be represented as a sum of these basis spectra, where THE CONTRIBUTION OF each is proportional to the fraction of the corresponding secondary structure type in that protein.

This approach typically yields results that are in good agreement with X-ray crystallography data. As a rule, the content of α-helices—which exhibit more pronounced spectral features in circular dichroism—is the easiest to predict, whereas determining the content of β-sheet elements and β-turns is somewhat more challenging. However, the primary advantage of the method lies not in determining the absolute proportions of individual secondary structure types, but in its exceptional value for observing how environmental changes affect the secondary—and thus tertiary—structure of a protein. The method is indispensable for investigating Protein Denaturation and characterizing protein fragments and Fibrous proteins.