Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin, I. P. 1968

Spatial Organization of the Protein Molecule
Methods for Studying the Secondary Structure of Proteins and Polypeptides
Electron Microscopy

Determining the size and shape of a protein or polypeptide molecule through direct observation offers undeniable advantages. Because Biopolymers are too small to be observed under a conventional Light Microscope, an Electron microscope must be used, in which ordinary light is replaced by a beam of electrons focused by electromagnetic lenses. Theoretically, the extremely short wavelength of electron beams should provide exceptionally high resolution—on the order of 0.005 Å. However, various technical difficulties significantly reduce the sensitivity of the method, yielding an effective resolution of about 5–10 Å for the best modern electron microscopes. This is quite sufficient to estimate the size and shape of a protein molecule or to visualize the a-helix of a synthetic polypeptide.

Unlike a conventional light microscope, where image contrast is determined by differences in the refractive indices of various PARTS OF THE object, in an electron microscope image contrast depends on the mass density of the object. Since protein and polypeptide particles are not dense enough on their own to scatter the electron beam effectively and thus lack sufficient contrast, it is often necessary to employ impregnation Methods (such as osmium impregnation), staining, or metal shadowing to enhance contrast. The latter two techniques are used most frequently.

Phosphotungstic acid is frequently used as a stain that is impermeable to electrons and selectively binds to Proteins. In this case, researchers often resort to so-called negative staining (where the excess stain is not completely washed away). As a result, the light-colored images of protein molecules stand out clearly against the dark Background of the unwashed acid.

The shadowing technique involves depositing a thin layer of metal onto the studied objects via vacuum evaporation. The evaporation is performed at a very shallow angle from a metal filament located at a relatively large distance from the object. A layer of metal is deposited on the side of the object's surface irregularities facing the filament, casting a shadow on the opposite side. The length of the resulting shadows allows researchers to estimate the dimensions of various surface protrusions. When such preparations are examined under an electron microscope, they yield images with very high contrast, significantly improving resolution and visibility down to particles 40 Å in diameter (or up to 15 Å in the case of fibers).

Using these techniques, researchers have recently succeeded in photographing individual a-helix macromolecules. Such images have been obtained for Polyglutamic acid, poly-y-benzoylglutamate, and other Polypeptides. For instance, microphotographs of poly-y-benzoylglutamate revealed rod-like particles measuring approximately 2030 Å. Given that the y-benzoylglutamate polypeptides had a Molecular Weight of about 350,000 with an amino acid residue weight of roughly 220, such a polypeptide consisted of approximately 1,500 residues. Consequently, each amino acid residue accounted for about 1.3 Å along the helix axis, which is quite close to the theoretical value (1.5 Å, According to the Pauling–Corey model). When Solvents that disrupt a-helices are used, microphotographs reveal patterns of random coils. When investigating Water-soluble polypeptides with ionizable side chains (such as polyglutamic acid or polylysine), one can also observe the melting process of the a-helix upon ionization of the side chains. In addition to polypeptide research, Electron Cell/15.html">Microscopy is widely used to determine the size and shape of Viruses and Fibrous proteins such as Collagen and fibrin. However, due to the technical limitations of electron microscopy, proteins cannot be studied in their natural hydrated state. Furthermore, drying proteins can introduce artifacts resulting from the aggregation or fragmentation of biopolymer molecules. Finally, this method is generally difficult to apply to The Study of biopolymers with a molecular weight of less than 10⁵.

Table 4. Degree of helicity of various proteins (in %)

When reviewing the aforementioned methods, it becomes evident that each has certain limitations. For instance, measuring optical activity does not yet provide rigorous proof for the existence of a-helices; Light absorption at 190 mµ by side-chain radicals reduces the accuracy of ultraviolet spectroscopy; and drying samples for electron microscopy introduces various artifacts, and so on. Naturally, all this warrants a certain degree of caution regarding data obtained by any single method and necessitates The Use of multiple independent techniques when studying the Introduction/11.html">Secondary Structure of a protein or polypeptide. For A number of proteins, results obtained using different methods often show good agreement, whereas in some cases they are highly contradictory (Table 4). This is particularly evident in the case of ribonuclease.

Despite the respective limitations of each method, all of them find wide application in the Study of the secondary structure of proteins and polypeptides. The relative simplicity of these methods, the feasibility of using several of them to estimate the degree of protein helicity in solution, and The ability to evaluate the type of secondary structure make them indispensable sources of information regarding the architecture of most proteins.