Fundamentals of Molecular Biology. Part 1: Molecular Cell Biology - A. N. Ogurtsov 2011

Nucleic Acids and Proteins
Hierarchy of Protein Structures

5.5.1. Primary Cell/13.html">Protein Structure. The Introduction/19.html">Primary Structure of a protein (Figure 101(a)) refers to the linear sequence of amino acid residues within its polypeptide chain.

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Figure 101 - Organization of protein structures: a - primary structure of a protein chain; b - a-helix; c - ß-Structure

Protein macromolecules consist of one or more polypeptide chains built from amino acid residues. One end of the chain, the N-terminus, features an NH₂ group, while the other end, the C-terminus, contains a COOH group. Amino Acids are linked together into Polypeptides via covalent peptide bonds.

The peptide bond is rigid, with the four atoms forming it (H-N-C=0) lying in the same plane (Figure 102).

Figure 102 - The peptide bond. Interatomic distances are given in angstroms

The electronic STRUCTURE OF THE peptide bond exhibits Resonance characteristics that span all four atoms of the peptide group, conferring rigidity as if all bonds possessed double-bond properties.

Consequently, the polypeptide chain can rotate relatively freely only around the a-carbon bonds designated as "A" and "B" in Figure 103.

Figure 103 - Resonance in the peptide bond

Due to The formation of the peptide bond, the H-N group acts as a potential Hydrogen bond donor, whereas the 0=C group serves as its acceptor. This process can be intuitively visualized as the "transfer" of an electron from nitrogen to oxygen.

5.5.2. Secondary Protein Structure. Secondary structure refers to the ordered conformation of polypeptide chains stabilized by Hydrogen Bonds between the C=0 and N-H groups of different amino acids.

The rigidity of inter-amino-acid peptide bonds and topological differences in the Structure of Amino acid side chains impose specific steric constraints on the Secondary structure of the polypeptide chain.

The torsion angles of rotation around the N-Cа and Са—С bonds of the main chain, illustrated in Figure 104(a), are designated as φ and ψ angles, respectively.

Figure 104 - Conformations of the polypeptide chain: a - the main polypeptide chain with a Cysteine side group; b - Ramachandran plot. Color intensity is proportional to the "allowedness" of the corresponding combination of backbone torsion angles φ and ψ (i.e., the depth of the energy "valley")

The arrows in the figure indicate the direction of rotation that increases the dihedral angle of the part of the chain closer to the viewer relative to its more distant segment.

To describe the stereochemistry of amino acid residues in Proteins, researchers use so-called Ramachandran plots—geodesic maps illustrating "allowed" and "forbidden" combinations of φ and ψ angles for specific protein conformations (Figures 104 and 105).

An example of such a mapping is shown in Figure 104(6). Without steric constraints on the geometry of the polypeptide chain, the torsion angles φ and ψ could take any arbitrary values. In that case, the point corresponding to a given amino acid residue's conformation could fall anywhere on the Ramachandran map.

Figure 105 - Steric constraints in the polypeptide chain: a - "allowed" conformations; b - "forbidden" conformation. 1 - φ = -90°, ψ = 120°; 2 - φ = -90°, ψ = -60°; 3 - φ = +90°, ψ = -90°

In reality, not all combinations of φ and ψ angles are permissible. This is because altering both angles inevitably shifts the relative positions of atoms in adjacent amino acid residues.

The close spatial approach of atoms—particularly bulky ones (such as oxygen, carbon, and nitrogen)—results in mutual repulsion. Overcoming this repulsion requires a significant energy input, rendering such polypeptide configurations unstable and their corresponding conformations "forbidden".

Steric constraints—the fundamental physical principle that two atoms cannot occupy the same space at the same time—significantly restrict the Number of viable combinations for the torsion angles φ and ψ.

Figure 105(a) shows Examples of "allowed" conformations corresponding to deep "valleys" on the Ramachandran plot, whereas Figure 105(b) illustrates an energetically unfavorable "forbidden" conformation corresponding to a "plateau" on the Ramachandran map.

The periodic alternation of peptide bonds largely determines the geometry of the resulting protein structures. Specifically, the periodicity of peptide bonds within The amino acid chain, along with the outward orientation of hydrogen and oxygen atoms from the protein backbone, restricts the existence of stable protein conformations to only a limited number.

In particular, the two conformations shown in Figure 101 are stable. They combine a minimized chain length with a maximized number of hydrogen bonds between atoms of different peptide groups.

The first conformation is the a-helix (Figures 101(6), 106, and 108(a)). The chain is coiled into a helix such that each peptide group oxygen forms a hydrogen bond with the hydrogen atom of a peptide group located Three amino acids away along the sequence (Figures 106 and 108(a)).

The Ramachandran plot for a-helices is shown in Figure 107(a).

The second conformation is the ß-structure, formed by multiple parallel chains (Figure 101(b)). Each chain is fully extended, and the overall Stability of the ß-structure is maintained by interchain hydrogen bonds, which can form either parallel or antiparallel arrangements (Figure 108(b,c)).

Figure 106 - Hydrogen bonds in the a-structure

ß-Structures can interact with one another to form entire protein sheets. The Ramachandran plot for ß-structures is presented in Figure 49(6). Ramachandran plots allow for the existence of Two Types of a-helices: right-handed (which absolutely dominate in proteins) and left-handed (which are virtually nonexistent).

Figure 107 - Ramachandran plots: a - for a-helices; b - for ß-structures. Numbers indicate minima: 1 - right-handed a-helix (primary); 2 - left-handed a-helix (rare); 3 - ß-structure

Left-handed helices are energetically less favored due to additional steric clashes caused by the presence of amino acid L-side chains.

Figure 108 - Secondary protein structure: a - a-helix; b - parallel ß-structure; c - antiparallel ß-structure

a-Helices form relatively rigid cylindrical structures. In diagrams, a-helices are depicted either as helical ribbons or cylinders, whereas ß-structures are represented as flat arrows or strips (Figure 109).

Figure 109 - Schematic representation of secondary structures: a - a-helices; b - ß-structures

Supersecondary structures (or elementary motifs) are also distinguished—these are thermodynamically or kinetically stable complexes of a-helices and ß-structures. Examples found in Globular proteins include (ßxß) elements (two parallel ß-strands connected by an "x" segment), the Rossmann fold (a ßaßaß element formed by two a-helix segments inserted between three parallel ß-strands), and the ß-meander (a sheet consisting of three or more antiparallel ß-strands; "meander" refers to a winding river in Greece).

5.5.3. Tertiary Protein Structure. The tertiary structure forms The basis of protein functionality, which requires precise Spatial Organization of large ensembles of amino acids.

Tertiary structure refers to the three-dimensional arrangement of all atoms in a protein molecule.

The stability of tertiary structure is driven by four types of interactions between side chains.

1. Covalent bonds between two cysteine residues (disulfide bridges) (Figure 110).

2. Electrostatic (ionic) interactions between oppositely charged amino acid residues (three positively charged and two negatively charged side chains). For example, the positively charged amino group of Lysine (NH₃⁺) is attracted to the negatively charged carboxyl group (COO-) of glutamic or aspartic acid.

3. Hydrogen bonds involving all amino acids that possess hydroxyl, amide, or carboxyl groups.

4. Hydrophobic interactions between nonpolar side chains and the aqueous environment.

Figure 110 - Formation of a disulfide bridge

Certain combinations of a- and ß-structures that ensure the functional Specificity of proteins are shown in Figure 111.

The tertiary structure is completely determined by the primary structure.

The key driving forces in the formation of tertiary structure are hydrophobic interactions, owing to their non-specific nature and Abundance.

A Hydrophobic core exists in most proteins. A decisive role in stabilizing the tertiary structure is played by the increase in the Entropy of Water (the solvent) during globule folding.

Figure 111 - Combinations of a-helices, ß-structures, and connecting protein strands determine protein topology: (1-5) - combinations of a-helices; (6-15) - combinations of ß-structures; (16-20) - a/ß combined protein tertiary structures

The characteristic way proteins organize their Spatial Structure—forming a hydrophobic core and a mosaic surface containing both hydrophilic and hydrophobic elements—limits the size of the globule, since as its volume increases, it becomes more difficult to form a strictly hydrophobic core. Starting at a molecular mass of approximately 14-16 kDa, the polypeptide chain forms two (or more) domains.

Domains are defined as regions within a protein's tertiary structure that possess a certain degree of structural autonomy. This structural autonomy is frequently complemented by functional autonomy. For example, in Enzymes, catalytic domains are often structurally segregated from regulatory domains.

The presence of domains provides the structural preconditions for greater internal flexibility and dynamics of protein molecules, achieved through the relative displacement of domains.

5.5.4. Quaternary Protein Structure. Quaternary protein structure refers to the aggregation of two or more polypeptide chains possessing tertiary structure into an oligomeric, functionally significant assembly.

The bonds that form and maintain The quaternary structure are identical to those involved in tertiary structure formation, with the exception of hydrophobic interactions.

Approximately half of all proteins exhibit a quaternary structure, including Hemoglobin, IMMUNOGLOBULINS, and Insulin. Nearly all DNA and RNA polymerases have a quaternary structure, as do numerous enzymes. Examples of proteins with quaternary structure include globular Actin (G-actin, Figure 22) and tubulin (Figure 26).

Main Functions of the quaternary protein structure.

1. Integration of multiple interrelated functions within a single structure. For example, the enzyme protein kinase A (see section 6.6.4) consists of two subunits: one catalyzes The transfer of a phosphate from ATP to a protein, while the other serves a regulatory function.

2. Architectural function. In the same protein kinase A, the substrate-binding site is formed by two domains that move closer together upon interaction with the substrate's phosphate group, shielding it from the surrounding solvent so that the reaction proceeds in its complete absence.

3. Facilitation of multiple interactions between the protein and extended structures. Owing to its quaternary structure, immunoglobulins combine two identical antigen-binding sites within a single molecule. The cooperativity of interaction between such sites and macromolecular Antigens, such as bacterial cell walls, makes antigen-immunoglobulin complexes significantly more stable than would be observed for a monomeric protein.

4. Regulatory function. The primary functional feature of quaternary structure—and presumably the rationale for its existence—is that the relatively weak interactions between subunits, The Nature of which heavily depends on the Tertiary Structure of each, are particularly well-suited for regulatory control and the modulation of protein activity. Changes in the tertiary structure of any given domain, induced by its interaction with a substrate or another Ligand, are transmitted via the relative weakness of interdomain contacts to its contact zone with another domain, altering the nature of that zone. Such a modification triggers a reorganization of the entire quaternary structure, thereby ensuring the transmission of the effect from one domain to others.

Thus, the hierarchy of protein structures can be represented schematically as shown in Figure 112.

Figure 112 - Hierarchy of protein structures: a - primary structure; 1 - amino acids; b - secondary structures; c - tertiary structure; d - quaternary structure