The Peptide Bond
When two amino acids condense to form a covalent peptide bond, a water molecule is removed; hydrogen and oxygen are lost from the carboxyl group (COOH), while the other amino acid loses hydrogen from its amino group (NH2). This condensation reaction results in the formation of an amide (R−CO−NH−R) bond. Once the amide forms, the lone pair of electrons from the nitrogen delocalize, allowing resonance which provides the nitrogen group a partial double-bond character forming a rigid, planar bond that can either be in the cis or trans configuration. During protein biosynthesis, the configuration most often adapted is the trans (1000:1) form over cis; however, isomerization between cis and trans occurs when the protein is unfolded or denatured. It is essential to understand the rigid character of the peptide bond because it limits rotation (flexibility) to the central carbon attached to the side chain, which greatly reduces the number of conformations the protein can adopt and is essential in forming secondary protein structures.
Primary (1o) Structure of Proteins
The primary protein structure was first postulated as a linear chain of amino acids in 1902. The primary structure of proteins is the sequence of alpha-amino acids covalently bound via amide bonds along the polypeptide chain, determined by the DNA of the gene that encodes for that protein. The number (length) and type of amino acids determine the molecular weight of the protein, with most between 20 and 100 kDa. A similarity in the composition does not imply a similarity in the sequence; likewise, similarities in the sequence do not necessarily imply a similar function. Convention dictates that the primary structure of a protein starts from the amino-terminal (N) and ends at the carboxyl-terminal (C). In most biological systems, protein synthesis occurs during translation in the ribosomes.
The linear unbranched protein polymers of amino acids may undergo posttranslational modifications, including acetylation, phosphorylation, glucosylation, and deamination. Alterations made to the order of the DNA sequence may change the amino acid sequence affecting the overall protein structure and function. To illustrate, if hemoglobin, the protein carrier of oxygen in the blood, replaces the 6th amino acid, glutamic acid, with valine, sickle cell anemia results from that single amino acid in a chain containing ~600 amino acids distorting disc-shaped red blood cells into crescent shapes (sickled) cells. To an extent, the polypeptide primary sequence influences chain folding, giving rise to the secondary and tertiary protein structures.
Secondary (2o) Structure of Proteins
During the ribosomal synthesis of proteins, folding is controlled by forming a molten globule state, semi-organized in-between structures with secondary structures formed but no fixed tertiary structure. From the molten globule, the protein further folds into several sequential intermediate states until the native structure is formed and typically has the lowest free energy state (tertiary (3o) structure). Exact folding is needed for enzymatic proteins as their functionality is derived from a cluster of functional amino acids that are not sequential (see catalytic triad below).
The secondary protein structure describes the local conformation of a segment of the polypeptide chain due to non-covalent hydrogen bonding interactions between atoms N-H and C=O groups along the polypeptide chain. These non-covalent interactions are highly repetitive due to the rigid planar amide bonds occurring along the single polypeptide backbone, not the amino acid side chains. The most common protein secondary structures are beta-sheets, helices, and short segments comprising the various turns, loops and random coils. Helices are the most common secondary structures, with the alpha-helix being the most prevalent. Numerous helical structures have different residues per helical turn, creating different pitches and angles, characterized by an average number of residues per helical turn and the number of atoms it takes. Thus, the 310 helix has 3 amino acids per turn containing 10 atoms. The alpha-helix (3.613) contains an average of 3.6 amino acids per turn containing 13 atoms. And the pi-helix has 4.1 amino acids per turn and 7-10 atoms in each turn. The amino acids of alpha-helices are sequential, while beta-sheets occur between sequential segments separated by short amino acid segments, often containing a turn of various sizes. Beta-sheets form hydrogen bonds between the carbonyl O of one amino acid and the amino H of another and are characterized if the pleated segments are parallel or antiparallel. Secondary structures are key to remember: the non-covalent hydrogen bonding interactions that stabilize and form these structures occur along the peptide backbone, relying on carbonyl and amino groups of different amino acids.
Secondary (2o) Structure of Proteins-The alpha-helix
The alpha-helix is the most common protein secondary structure. Also termed the 3.613-helix contains an average of 3.6 amino acids per turn containing 13 atoms. Within an alpha-helix, every amino acid forms a hydrogen bond to an amino acid (i+4) away, thus the 1st amino acid hydrogen bonds to the 5th amino acid, the 2nd amino acid hydrogen bonds with 6th, and the 3rd H-bonds to the 7th, and so on until the end of the helical structure. This helical structure exposes the R-side chains outward radially from the helix, and while R-groups do not play a role in stabilizing the helix, they are essential in determining how secondary elements fold into ternary structures. Amino acids commonly occurring in helix include amino acids with linear side chains, such as Methionine, Alanine, Leucine, Glutamic Acid, Lysine, Arginine, Glutamine, and Histidine. In the alpha-helix, each amino acid has a rise of 1.5 Å and a 100o rotation which leads to 3.6 amino acids per twist and a 100o rotation per amino acid, making two adjacent amino acids on opposite sides of the helix. The α-helix is highly stable due to numerous hydrogen bonds between the amino acids C=O and N-H groups (C=O—H-N).
Secondary (2o) Structure of Proteins-The beta-sheet
Beta-sheets consist of beta strands (β-strands) of 3 to 10 amino acids connected sequentially, forming >2 or more (typical 5 to 15 amino acids) backbone hydrogen bonds between strands, resulting in a twisted, pleated sheet. β-strands interact via hydrogen bonding to form a β-pleated sheet. Like the alpha-helix, beta-sheets form hydrogen bonds between the amino acids C=O and N-H groups (C=O—H-N) along the peptide backbone. The β-strands can either orient a parallel, where all strands have the same biological C->N orientation causing hydrogen bonds to form on an angle.
Antiparallel, which alternates between C->N and N->C along the stretches of a polypeptide chain resulting in a long extended strand which allows hydrogen bonding to occur at minimal angles to each other. Beta-sheets alternate side-R-groups above and below the pleated sheet where large bulky side chains, including aromatic (Tyr, Phe, Trp), branched (Thr, Ile, Val), and large sulfur-containing (Cys, His) tend to be found.
Secondary (2o) Structure of Proteins-The turns, Loops and Random Coils
Turns along protein chains are essential for proper tertiary folding and alignment of pleated beta-strands to form antiparallel beta-sheets. Turns occur when the protein chain needs to change direction to connect two other secondary structure elements and stabilize by a single hydrogen bond along the peptide backbone.
The beta-turn is the most common, where a directional change occurs over four amino acid residues. Proline is common in loop sequences as its molecular configuration does not allow the formation of other helical or beta-sheet secondary structures. Proline disrupts α-helices and β-sheets as it has no hydrogen atom at the α-NH2 for H-bonding, which is essential in stabilizing secondary structures; therefore, proline is restricted to the first residue of an α-helix & at the edge strands of β-sheets. Another common amino acid in ‘turn’ sequences is glycine, which has no side chain carbons to prevent water from hydrogen bonding with the peptide backbone, which breaks α-helices. Glycine also lacks a bulky sidechain to drive β-sheets, therefore, is restricted primarily to loop/turn sequences. Turns, loops and linkers are essential for the protein to adapt the correct tertiary structure.
Some protein segments do not form a regular secondary structure or repeating hydrogen bonding pattern along the peptide backbone; these regions are random coils found at the protein’s N-terminus and C-terminus and within loops. Loops are any unstructured regions between regular secondary structure elements. Since loops are found at the terminus of the protein, they are often exposed to solvent, and amino acids are polar or charged.
Tertiary (3o) Structure of Proteins
The tertiary protein structure is formed by folding the entire polypeptide chain, which is highly dependent on the environment. The linear protein chains fold into highly-specific 3D conformations, affected by the type and sequence of amino acids and resultant secondary structures. Tertiary structures range from highly fibrous (collagen, actomyosin, fibroin (silk) and keratins (hair and wool)) to globular (insulin, hemoglobin, ovalbumin (egg protein)) and affect their function. Fibrous proteins tend to be structural, while globular proteins are likely enzymes.
In aqueous environments, proteins fold, positioning hydrophobic residues toward the interior and the hydrophilic amino acids to the exterior exposed to water (hydrophobic effect). Covalent and non-covalent interactions between amino acid side chains (R-groups) form to maintain the tertiary structure and result from the proximity between them. For example, a covalent disulfide bridge may form between cysteine residues apart in sequence but close in 3D proximity, providing rigidity for the resulting 3D structure. Non-covalent interactions between amino acids R-groups stabilize and maintain the tertiary structure and are dependent on pH. Non-covalent interactions by decreasing strength include charge-charge, charge-dipole, hydrogen, dipole-dipole, and induced dipole-charge interactions. London dispersion forces and pi-pi stacking are weaker but still relevant forces.
When the non-covalent forces, either along the backbone or located on side changes, are disrupted, changes in tertiary and secondary result in a loss of native structure and the protein is denatured. Protein denaturation results in loss of function for enzymes, alterations in their solubility (collagen to gelatin), and the physiochemical properties (kneading flour and water to make a viscoelastic dough for bread) they confer to foods. Heat, pH, ionic strength and type, and solvent environment denature proteins causing a loss in native 2o and 3o, possibly 4o when present; however, 1o structure remains unaffected unless amide bonds are hydrolyzed.
Tertiary (3o) Structure of Proteins-The Catalytic Triad
The catalytic triad arises from the tertiary protein structure and is the most important functional element of digestive enzymes as it adds water during hydrolysis reactions breaking macromolecules into smaller functional units that can be transported across the epithelial layer. During digestion, enzyme hydrolysis reactions contain the three amino acids catalytic triad with aspartate, histidine, and serine. Although all digestive enzymes contain these three amino acids, their location in the primary protein sequence differs, and their proximity to each other depends on the protein folding of their secondary and ternary structures.
Aspartate for lingual lipase is at position 203, while for gastric lipase, it is at 324, and finally, for pancreatic lipase, it is at 177. Histidine for these three enzymes is located at 257 and 353 or 264. The location of serine is 144 for lingual lipase and 153 for both gastric and pancreatic lipases. The key to the catalytic triad is to overcome the fact that no amino acids are strong nucleophiles; hence the base in the catalytic triad polarizes and deprotonates the nucleophile increasing its reactivity to form a covalent bond with the substrates’ carbonyl group. The amino acid aspartate in the catalytic triad operates by first deprotonating, initiating a charge relay sequence that polarizes and activates the nucleophile serine relying on the base histidine as an intermediate to relay charge. Once the nucleophile, serine, is activated, it forms an intermediate covalent bond with the substrate and, using water, hydrolyzes peptide, alpha-1-6 glycosidic, or ester bonds, depending on the enzyme.
Quaternary (4o) Structure of Proteins
Primary, secondary, and tertiary structures are present for all proteins, however not all proteins have quaternary (4o); when more the one protein forms a functional unit, a structure such as hemoglobin (4 myoglobin protein unit) to hold iron, allowing oxygen to circulate in the blood, or DNA polymerase (2 protein unit) to transcript DNA to produce the proteins corresponding to the sequence of DNA. These levels of structure are important for the color of meat, especially red meat, when fresh (red or brown), cured (pink) or cooked (brown). It is also key to the pigmentation of meat mimics without using betalains (pigment from beets).
