Peptide Bond Formation and Primary Protein Structure
Proteins are polymers of amino acids that fold to shapes thatconfer function on the proteins. In biological systems, the amino acids are linked together by a large ribonucleic acid/protein nanoparticle called the ribosome. Thus, as the amino acids are linked together to form a specific protein, they are placed within a very specific order that is dictated by the genetic information contained within the a specific type of RNA called messenger RNA (mRNA). The mRNA sequences is encoded in the genomic DNA sequence. Thespecific ordering of amino acids is known as the protein's primary sequence. The translation mechanism used by the ribosome to synthesize proteins will be discussed in detail in Chapter XX.
The amino acids are linked together using dehydration synthesis (loss of water) reaction that combine the carboxylic acid of the upstream amino acid with the amine functional group of the downstream amino acid to form an amide linkage (Figure 2.10). Note that the reverse reaction is hydrolysis and requires the incorporation of a water molecule to separate two amino acids and break the amide bond. Notably, the ribosome serves as the enzyme that mediates the dehydration synthesis reactions required to build protein molecules, whereas a class of enzymes called proteases are required for protein hydrolysis.
Within protein structures, the amide linkage between amino acids is known as the peptide bond. Subsequent amino acids will be added onto the carboxylic acid terminal of the growing structure. Proteins are always synthesized in a directional manner starting with the amine and ending with the carboxylic acid tail. New amino acids are always added onto the carboxylic acid tail, never onto the amine of the first amino acid in the chain. The directionality of protein synthesis is dictated by the ribosome and is known as N- to C- synthesis.
The figure below shows a generic structure from a longer peptide or protein.
Proteins range in size from around 50 amino acids in length to the largest known protein, titin (aka called connectin), a muscle protein. The human version has over 34,000 amino acids and a molecular weight over 3.9 million! Some consider structures with fewer than 50 amino acids to be peptides (Figure 2.13). Others suggest that structures with 40-50 amino acids should be considered small proteins. One way to differentiate them is by how they are synthesized in vivo. Storz et al consider polypeptides to be small proteins if they encoded in the genome by a continuous stretch of DNA base in an "open reading frame" (doi: 10.1146/annurev-biochem-070611-102400). Peptides, on the other hand, could be considered those that are:
- "intrinsically disordered" with no definite fold,
- derived from proteins by proteolyis, and/or
- not synthesized by ribosomes
As genomes were sequences and annotated, some arbitrary parameter were set. For the yeast genome, annotation for proteins were defined if open reading frame lead to a polypeptide of 100 amino acids or a molecular weight of 11,000 (average molecular weight of an amino acid is 110). If no cutoff was used, the number of proteins encoded by the genome would be huge. Submissions of DNA sequences to the NIH GenBank must encode proteins no smaller than about 66 amino acids (MW about 7250). Even this ignores small proteins that have been isolated and characterized from cells. So the cutoff of 50 amino acids (MW about 5500) derived from open reading frames seems like the best arbitrary cutoff going from peptides to proteins.
Due to the large pool of amino acids that can be incorporated at each position within the protein, there are billions of different possible protein combinations that can be used to create novel protein structures! For example, think about a tripeptide made from this amino acid pool. At each position there are 20 different options that can be incorporated. Thus, the total number of resulting tripeptides possible would be 20 X 20 X 20 or 203, which equals 8,000 different tripeptide options! Now think about how many options there would be for a small peptide containing 40 amino acids. There would be 2040 options, or a mind boggling 1.09 X 1052 potential sequence options! Each of these options would vary in the overall protein shape, as the nature of the amino acid side chains helps to determine the interaction of the protein with the other residues in the protein itself and with its surrounding environment.
Nearly 200,000 3D structure of biomacromolecules are known. How can we simplify our understanding of the diversity of protein structure. Is each structure completely unique? What do they have in common?
To simplify and inform our understanding of the diversity of biological organisms, we place them into groups (from domains and kingdoms to genus and species), based on common characteristics. We likewise, proteins are divided into a hierarchy of structures with increasing information content. This overview describes the four basic levels of protein structure, primary (10), secondary (20), tertiary (30) and quaternary (40). Each will be probed in greater deal in the next chapter. These classes of structures will be illustrated below with a protein named hydroxynitrile lyase (5Y02). (This protein has been simplified to illustrate key features of structure as will be described at the end of these description).
Primary (10) Structure: the amino acid sequence of a protein.
The primary (10) structure of a protein is simply the amino acid sequence of a protein written from N- to C-terminal. It does not require visualization to describe it. Consider two different short continuous sequences from the hydroxynitirle lyase:
- Gln-Lys-Gln-Ile-Asp-Gln-Ile or in single letter code QKQIDQI. This is the sequence for amino acids 20-26 in the protein. This stretch of 10 structure has multiple repeated amino acids.
- Asp-Leu-Gly-Pro-Ala-Val or in single letter code DLGPAV. This is the sequence for amino acids 48-53 in the protein. This stretch of 10 structure does not contain repetitive amino acids.
A 2-D line drawing of the sequence offers more information about the sequence but does not provids information about the actual conformation of these sections of 10 structure within a given protein. These can be shown in the figure below in which the overall structure of the protein is shown in grey sticks with the short stretches of primary structure shown in colored spacefill and 2D line drawings.
Secondary (20) and Tertiary (30) Structures
Secondary (20) structures are repetitive structures with a protein held together by hydrogen bonds between amide Hs and carbonyl Os in the backbone main chain atoms. It's most easily examined through specific rendering of the overall tertiary (30) or 3-D structure of the protein. Five different renderings showing the 3D (the tertiary structure of the protein are shown below.
Representation A shows a stick drawing of the protein with red indicating bonds to oxygen and blue bonds to nitrogen. No bonds to hydrogen are shown as these are too small to be detected using common techniques used to determine structures of such large molecules. It looks like a complicated mess of bonds so understanding unique features with the 30 structure of the protein is difficult. Representation B shows just the backbone of the protein. The outline of how the protein twists and turns in space becomes more evident. The N- and C-terminal ends are more clearly seen.
Representation C shows just the bonds connecting the alpha C atoms of each amino acid. The protein's overall topology is now clearly evident. If you follow the chain from the N- to C-terminal ends, it should be evident that there are regularities in the conformations of the protein chain. The individual yellow zig zags are called beta strands. These strands appear elongated and aligned with other beta strands to form a larger beta sheet. The sheet is held together through hydrogen bonds between backbone amide Hs and carbonyl Os on adjacent strands. Beta strands are a type of secondary structure.
The red zig-zag lines represent another type of secondary structure called the alpha-helix. The helix is again held together by hydrogen bonds between amide Hs and carbonyl Os within a single continuous strand. The backbone of the alpha helix appear less elongated than in a beta strand as it is wound into a coil (the alpha helix) along a central axis. If you took tweezers (using atomic force microscopy) and pulled on the helix, it could stretch and become more elongated like the beta strands.
The rest of the protein alpha carbon chain shown in blue is less regular. However, it is still ordered as it propagates through space in what is termed a random coil. In the protein it adopts mostly a fixed conformation but it has more conformational flexibility than alpha helices and beta sheets. The alpha helices and beta strands (sheets) are examples of secondary structure.
Representations D and E are cartoon drawings clearly showing the alpha helices (red) and beta stands and sheets (yellow). It would be extremely difficult to discern alpha or beta secondary structures with a stick representations showing all the bonds in a protein. Some of the atoms must be visually (not literally) removed to see the repetitive propagation of the protein backbone through overall structure. If your goal was to understand the disposition of side chains in a small part of a protein, a cartoon view by itself would not be useful. Modeling programs allow mixed rending of a protein to include both cartoon and stick representations together.
Secondary structures, held together by hydrogen bonds between backbone atoms are characterized by repetitive changes in the angle of propagation of the chain between connected amino acids in an alpha helix and strand. In a give beta strand, the relative change in angle of propagation is nearly 00 compared to a much large angular change required to bend the amino acid backbone into an alpha helix. Here is the IUPAC definition of secondary structure. We added the word "repetitive" to clear show that random coils are not an example of secondary structure.
Definition: Secondary Structure (from the IUPAC Gold Book)
The [repetitive] conformational arrangement (α-helix, β-pleated sheet, etc.) of the backbone segments of a macromolecule such as a polypeptide chain of a protein without regard to the conformation of the side chains or the relationship to other segments.
Separate protein chains often interact through noncovalent interactions and sometimes through disulfide bond formation between free cysteine side chains on different chains to form dimers, trimers, tetramers, octamers, etc. Dimers can be homodimers (if the two chains are identical) or heterodimers (if they are different). The example we used in the section, hydroxynitirle lyase, forms a homodimer, as shown in the figure below. The left images shows a cartoon version, with one monomer in orange and the other identical monomer in green. The images to the right shows a translucent surface representation of the dimer, with the car
The right hand mixed-rending image shows a translucent surface image of each monomer and underneath the cartoon image
- primary structure: the linear amino acid sequence of a protein
- secondary structure: regular repeating structures arising when hydrogen bonds between the peptide backbone amide hydrogens and carbonyl oxygens occur at regular intervals within a given linear sequence (strand) of a protein or between two adjacent strands
Disulfide bonds within individual chains and between them stabilize both tertiary and quaternary structures of both peptides and proteins. These are illustrated in the figure below.
Protein Shape and Function
The primary structure of each protein leads to the unique folding pattern that is characteristic for that specific protein. In summary, the primary sequence is the linear order of the amino acids as they are linked together in the protein chain (Figure 2.15). In the next section, we will discuss protein folding that gives rise to secondary, tertiary and sometimes quaternary protein structures.
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