3.2: Levels (Orders) of Protein Structure

The three levels of polypeptide structure are primary, secondary, and tertiary structure. Quaternary structure arises from an association of two or more polypeptides that create higher-order protein structures. Super imposed on these levels, or orders. are still other features of protein structure. These are created by the specific amino acid configurations in a mature, biologically active protein. Let’s begin with a look at primary structure.

3.2.1. Protein Primary Structure: L-Amino Acids and the -C-C-N- Polypeptide Backbone

The primary structure of a protein simply refers to the amino acid sequence of its polypeptide chain(s). Cells use only twenty amino acids to make polypeptides and proteins, although they do use a few additional amino acids for other purposes.

The peptide linkages between amino acids in polypeptides form in condensation reactions in cells during protein synthesis (i.e.,translation). The linkages involve multiple covalent bonds. They break and rearrange between the carboxyl and amino groups of amino acids during linkage formation. The twenty amino acids found in proteins are shown below in Figure 3.2.

Figure 3.2: Chemical characteristics of the twenty amino acids found in the proteins of cells. All side chains (R groups) are shown at the bottom of each structure, bound to an \(\alpha\) carbon

In all twenty amino acids (except glycine), the \(\alpha\)−carbon is bound to four different groups, making them chiral or optically active.

CHALLENGE

Circle the \(\alpha\)-carbon in each of the amino acids in Figure 3.2. Why is glycine not optically active?

3.2.1. a Peptide Bond Formation and Polypeptide Primary Structure

Recall that chiral carbons allow for mirror image D and L (or d and l) optical isomers. Remember, only the lower-case d and l define the optical properties of isomers. Just to make life interesting, L–amino acids are dextrorotary in a polarimeter, making them (d)–amino acids! While both (d)– and (l)–amino acid enantiomers exist in cells, only (d)–(i.e., L–) amino acids (along with glycine) are used by cells to build polypeptides and proteins by the process called translation.

We say that polypeptides have polarity because they have “free” carboxyl ends and free amino ends. Amino acid side chains end up alternating on opposite sides of a C-C-N-C-C-N-... polypeptide backbone because of covalent-bond angles along the backbone. You can prove this to yourself by making a short polypeptide with the kind of molecular modeling kit you may have used in a chemistry class! The C-C-N-C-C-N- backbone is the underlying basis of higher orders (or levels) of protein structure. A partial polypeptide is shown below in Figure 3.3.

Figure 3.3: The amino and carboxyl ends of a polypeptide define its polarity, with positively charged amino and negatively charged carboxyl ends at physiological pH. The positions of the circled amino acid R groups alternate along the polypeptide backbone. The two R groups in the carboxyl region (right) are hydrophobic; the two in the amine region (left) are polar.

132-2 Amino Acid Sequence & Protein Primary Structure

3.2.1.b Determining Protein Primary Structure - Polypeptide Sequencing

Primary structure is dictated directly by the gene encoding the protein. After transcription of a gene, a ribosome translates the resulting mRNA into a polypeptide. Frederick Sanger demonstrated the first practical protein sequencing method when he reported the amino-acid sequence of the two polypeptides of bovine (cow) insulin. His technique involves stepwise hydrolysis (called an Edman Degradation) of polypeptide fragments; each hydrolysis leaves behind a single amino acid that can be identified, and a polypeptide fragment shortened by one amino acid. Sanger received a Nobel Prize in 1958 for this feat. By convention, the display and counting of amino acids always starts at the amino end (N-terminus), the end with a free \(\rm NH_2-\)group (as suggested in Figure 3.3).

For some time now, the sequencing of DNA has replaced most direct protein sequencing. The method of DNA sequencing, colloquially referred to as the Sanger dideoxy method, quickly became widespread and was eventually automated, enabling rapid gene (and even whole-genome) sequencing. Now, instead of sequencing polypeptides directly, we can infer amino-acid sequences from gene sequences isolated by cloning or revealed after complete genome sequencing projects. This is the same Sanger who first sequenced proteins, and yes, he won a second Nobel Prize for the DNA sequencing work in 1980!

CHALLENGE

Both optical forms of amino acids exist in cells, but only the L-isomer occurs in biological proteins. When and why do you think that L-amino acid isomers were selected for use in proteins of living cells today?

The different physical and chemical properties of each amino acid result from the side chains on its \(\alpha\)−carbons. The unique physical and chemical properties of polypeptides and proteins are determined by their unique combination of amino acid side chains and their interactions within and between polypeptides. In this way, primary structure reflects the genetic underpinnings of polypeptide and protein function. The higher-order structures that account for the functional motifs and domains of a mature protein derive from its primary structure. Christian Anfinsen won a half share of the 1972 Nobel Prize in Chemistry for demonstrating that this was the case for the ribonuclease (RNAse) enzyme; Stanford Moore and William H.Stein earned their quarter shares of the prize for relating the structure of the active site of the enzyme to its tertiary structure and its catalytic function. See Anfinsen et al. 1972 Chemistry Nobel Prize for more.

3.2.2. Protein Secondary Structure

Secondary structure refers to highly regular local structures within a polypeptide (e.g., \(\textbf{\alpha}\)−helix) and either within or between polypeptides(\(\textbf{\beta}\)−pleated sheets). Linus Pauling and his coworkers suggested these two types of secondary structure in 1951. A little Linus Pauling history would be relevant here! By 1932 Pauling had developed his electronegativity scale of the elements, which could predict the strength of atomic bonds in molecules. He contributed much to our understanding of atomic orbitals and later of the structure of biological molecules. He earned the 1954 Nobel Prize in Chemistry for this work. He and his colleagues later discovered that sickle cell anemia was due to an abnormal hemoglobin. They went on to predict the \(\alpha\) helical and \(\beta\)−pleated sheet secondary structure of proteins (Figure 3.4, below).

Figure 3.4: Left: a computer-generated 3D image (left) shows \(\alpha\)-helical and pleated sheet “ribbon” regions separated by less-organized random-coils of amino acids in each of the two polypeptides (upper pink and lower green) of a two-subunit protein. The \(\beta\)-pleated sheet regions are shown as aligned arrow-ribbons. Right: Pleated sheet structures result when hydrogen bonds form between adjacent regions shown at the right.

Though Pauling did not earn a second Nobel for these novel studies of molecular genetics, he did win the 1962 Nobel Peace prize for convincing almost ten thousand scientists to petition the United Nations to vote to ban atmospheric nuclear bomb tests. More about his extraordinary life (e.g., at L. Pauling-a Short Biography) is worth a read! Coincidentally, Max F. Perutz and John C. Kendrew earned the 1962 Nobel Prize in Chemistry for their X-ray crystallographic studies of the 3D structure of hemoglobin. Clearly, 1962 was a good year for Nobel prizes for protein studies!

Secondary structure conformations occur due to the spontaneous formation of hydrogen bonds between amino groups and oxygens along the polypeptide backbone, as shown in the two left panels in Figure 3.4. Note that amino acid side chains play no significant role in secondary structure.The \(\alpha\) helix or \(\beta\) sheets are the most stable arrangement of H-bonds in the chain(s), and both are typically found in the same protein (on the right in Figure3.4). These regions of ordered secondary structure in a polypeptide can be separated by varying lengths of less-structured peptides called random coils.

All three of these secondary-structure features can occur in a single polypeptide or protein that has folded into its tertiary structure, as shown at the right in the illustration. The pleated sheets are shown as ribbons with arrowheads representing N-to-C or C-to-N polarity of the sheets. As you can see, a pair of peptide regions forming a pleated sheet may do so either in the parallel or antiparallel directions (look at the arrowheads of the ribbons), which will depend on other influences dictating protein folding to form tertiary structure.

Some polypeptides never go beyond their secondary structure, remaining fibrous and insoluble. Keratin is perhaps the best-known example of a fibrous protein, making up hair, fingernails, bird feathers, reptilian (but not fish!) scales, and even filaments of the cytoskeleton. However, most polypeptides and proteins do fold and assume tertiary structure, becoming soluble globular proteins.

133-2 Protein Secondary Structure

3.2.3. Protein Tertiary Structure

Polypeptides acquire their tertiary structure when hydrophobic and nonpolar interactions between amino acid side chains spontaneously draw them together to exclude water, aided by the formation of salt bridges and H-bonds between polar side chains in the interior of the globular polypeptide. In this way, \(\alpha\) helices or \(\beta\) sheets are folded and incorporated into globular shapes. Polar (hydrophilic) side chains that can find no other side-chain partners are typically found on the outer surface of the “globule,” where they interact with water and thus dissolve the protein (recall water of hydration).

Though based on noncovalent bonds, tertiary structures are nonetheless strong simply because of the large numbers of otherwise-weak interactions that form them. The forces that cooperate to form and stabilize 3D polypeptide and protein structures are illustrated below in Figure 3.5.

Figure 3.5: Tertiary structure is created by noncovalent hydrophobic (non-polar) amino acid interactions as well as H-bonding and ionic interactions in the interior of a polypeptide, leaving charged (hydrophilic) amino acid side chains to interact with water on the exterior of a typical “globular” protein. Covalent disulfide bonds between cysteine amino acids help stabilize tertiary structures.

Covalent disulfide bonds between cysteine amino acids in the polypeptide (shown in Figure 3.5) can further stabilize tertiary structure. Disulfide bonds (bridges) form in a polypeptide when cysteines far apart in its primary structure of the molecule end up near each other. Then the SH (sulfhydryl) groups in the cysteine side chains are oxidized, forming the disulfide (-S-S-) bonds (Figure 3.6).

Figure 3.6: Disulfide bonds (or bridges) form when cysteine amino acids in one or more polypeptides end up near each after it (or they) fold into higher-order structures. Oxidation reactions between the close SH (sulfhydryl) groups (left) result in linking their two sulfurs in a disulfide bond, or bridge (right).

134-2 Protein Tertiary Structure

135-2 Disulfide Bridges Stabilize \(3^0\) Structure

Disulfide bridges in a protein are crucial to the active protein. Imagine how changes in temperature or ionic strength would disrupt noncovalent bonds required for the correct 3D shape of the active protein. Unaffected by such changes, disulfide bridges limit the disruption and enable proteins to refold correctly when conditions return to normal (think homeostasis!)

CHALLENGE

Disulfide bonds are more common in blood proteins than in intracellular proteins. Since cytosol is typically a reducing environment, cysteines tend to be reduced (i.e., in the –SH state). What does this tell you about blood?

As we’ll see, protein activity can be regulated by chemical modification (e.g., phosphorylation).