Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Optical Properties of Proteins

One of the most important Optical Properties of Peptides and Proteins is the Rotation of the plane of light polarization, or optical activity. Optical activity consists of two components.

1. The first component is the sum of the increments of all asymmetric a-C atoms that make up the peptide chain. In a peptide chain, all a-C atoms typically rotate the plane of polarization to the left. In other words, free Amino Acids and amino acids incorporated into a peptide chain may rotate the plane of polarization in opposite directions. The reason is that both the direction and the angle of rotation by free amino acid molecules heavily depend on the state of the amino, carboxyl, and other dissociating groups in the side R-radical, the COMPOSITION OF THE solution, its pH, and certain other environmental factors. When a peptide chain is formed, uncharged CO-NH groups of the peptide bond are always located near the asymmetric C atom, which eliminates the complex influence of charges on the magnitude of the rotation angle. As a result, the increments for the a-C atoms of various amino acids level out (becoming practically identical). This effect does not occur with terminal amino acids, but THE CONTRIBUTION OF these two atoms to the total specific rotation of the entire peptide chain is extremely small.

In practice, when analyzing this issue, instead of the empirical specific optical activity [a], the so-called effective residue rotation [m] is used, expressed as follows:

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where M₀ is the Molecular Weight of the residue, and n is the refractive index of the medium.

Let us consider Examples for peptides that lack a well-organized Secondary Structure, i.e., exist in a statistical coil state. For instance, Polyglutamic acid in an alkaline medium (when it is fully ionized, carries a maximum negative charge, and thus lacks a helical conformation) has a value of [a]_D = -107∘ (specific activity measured at the sodium D-line, 589.3 mμ). The value of [m]_D at M₀=129 and n=1.37 is:

For polylysine in 6 N Hydrochloric acid, [а]_D=-80⁰, M₀=13°, n=1.38. Hence, [m]_D= -82°. As can be seen, the side-chain Structure of Amino acids does not exert a very strong influence on the effective angle of residue rotation. Similar fluctuations are observed in proteins as well. For denatured proteins, [m] ranges from -85 to -100°. These variations reflect differences in the Amino Acid Composition of the proteins.

2. The second component of protein optical activity is determined by The structure of the macromolecule as a whole. If an a-helix is the primary element of the Introduction/11.html">Secondary structure of a peptide chain, it should make a major contribution to the magnitude of optical activity, typically ranging from 80 to 100-105⁰. In the most general case, the backbone valence chain of a right-handed helix rotates the plane of polarization to the right, while that of a left-handed helix rotates it to the left. Since natural proteins, which consist of L-amino acids, form right-handed helices, they exhibit right-handed rotation. From all the above, a question naturally arises: can we rely on optical activity parameters to establish the very presence of a helical structure in a peptide chain, and can we, by performing appropriate calibration, estimate the degree of helicity of a peptide chain? An absolutely precise and definitive affirmative answer to these questions cannot yet be given. The only thing that can be stated with absolute certainty is that the ordering of globular protein macromolecules manifests as a large positive increment in optical activity.

A typical curve showing changes in optical activity during a process such as thermal Protein Denaturation is presented in Fig. 4.1.

The mere fact that an ordered structure breaks down, which is manifested by A change in optical activity, is not definitive proof of the presence of an a-helix specifically or of any helix at all. Such a Conclusion can only be drawn from the aggregate data of various Research Methods.

Fig. 4.1 Typical melting curve of Protein secondary structure