6: Amino Acids and Protein structures (2025)

Pre-Lecture

Structure of Amino Acids

Amino acids are the fundamental building blocks of proteins, each composed of a central (alpha) carbon atom bonded to four distinct groups: an amino group (-NH₂), a carboxyl group (-COOH), a hydrogen atom, and a variable R group or side chain that dictates the specific chemical properties of the amino acid. The amino group (-NH₂) is basic and acts as a hydrogen donor. It plays a critical role in peptide bond formation and enhances the solubility of amino acids in aqueous environments. The carboxyl group (-COOH), on the other hand, is acidic and functions as a hydrogen acceptor. It is essential for acid-base interactions and also participates in forming peptide bonds by reacting with the amino group of another amino acid. The central (alpha) carbon has a tetrahedral geometry, enabling the existence of stereoisomers—D and L forms—of amino acids. Among these, the L-forms are predominant in biological systems. Lastly, the R group (side chain) is the defining feature that varies between amino acids and determines their polarity, charge, reactivity, and role in protein folding and biochemical interactions.

• Amino Group (-NH2): Acts as a hydrogen donor and is basic.

• Carboxyl Group (-COOH): Acts as a hydrogen acceptor and is acidic.

• Central (Alpha) Carbon: Allows for tetrahedral configuration and forstereoisomerism, leading to D and L forms.L-forms are predominant.

• R Group (Side Chain):Defines the properties, including its polarity, charge, and participation in reactions.

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Effects of Temperature on Proteins

When proteins are chilled, such as when storing enzymes or other protein-based solutions in the refrigerator or on ice, the molecular movements slow down due to decreased kinetic energy. Imagine placing a bottle of milk or an egg white solution in the fridge—everything still looks the same, and the proteins in the solution are still intact. This is because cold temperatures help preserve the protein structure by preventing the breakdown of the bonds that maintain their shape. However, in some cases, proteins might aggregate—or clump together—when stored for too long or under certain buffer conditions. This clumping is usually reversible. For instance, some lab enzymes form a cloudy suspension when cold but regain clarity and function once gently warmed to room temperature. A practical example would be cold storage of insulin, which must be kept refrigerated to maintain its active structure, but must not be frozen, as freezing can cause irreversible aggregation.

Now compare this with what happens when you heat proteins. Think about cooking an egg: when you crack a raw egg into a hot frying pan, the egg white, which is mostly made of a protein called albumin, turns from clear and runny to white and solid. This change is a visible example of protein denaturation. As the temperature rises, the albumin proteins unfold (lose their secondary and tertiary structures) and then tangle together in a new, disordered way, which is what causes the egg white to firm up. These changes are irreversible—you cannot uncook an egg or restore the original structure of albumin. The same principle applies in biology: if human body temperature rises too high (such as in heat stroke or high fever), critical enzymes may denature, losing their shape and function, which can lead to cellular dysfunction and even death if not reversed quickly.

• Chilling Proteins: Cold temperatures slow down enzymatic activities and metabolic reactions but generally do not cause denaturation.

• Heating Proteins: High temperatures cause proteins to denature by breaking hydrogen bonds, and disrupting hydrophobic interactions, and altering the protein’s secondary, tertiary, and quaternary structures. This irreversible process results in the loss of biological function.

Amino Acid Structures and Classification

Each amino acid has a central carbon atom—called the alpha carbon—and this carbon is tetrahedral, meaning it forms four single bonds with four different groups: an amino group (-NH₂), a carboxyl group (-COOH), a hydrogen atom (H), and a variable R group (also called the side chain). Because all four groups are different, this carbon is chiral (except in glycine, where the R group is another hydrogen). This chirality means amino acids can exist in two mirror-image forms called D (dextro) and L (levo) isomers, similar to how your left and right hands are mirror images of each other but not superimposable. In nature, especially in proteins, almost all amino acids are found in the L-form. This is not just a coincidence—it’s because the enzymes that make proteins (like ribosomes during translation) and the enzymes that break down or modify proteins are designed to recognize only L-amino acids. This selective use of L-amino acids is a fundamental characteristic of life on Earth, and it helps maintain the proper structure and function of proteins.

Now, when amino acids come together to form proteins, they are connected by peptide bonds. A peptide bond is a special covalent bond that forms when the carboxyl group (-COOH) of one amino acid reacts with the amino group (-NH₂) of another. During this reaction, a molecule of water (H₂O) is removed—a process called dehydration synthesis—and the result is a strong bond linking the two amino acids together. This chain can grow longer and longer to form polypeptides, which fold into functional proteins.

Finally, the 20 natural amino acids that are used to build proteins can be grouped based on the characteristics of their R groups (side chains). These groups are: (1)Acidic (have a negatively charged carboxylic acid group, like glutamic acid and aspartic acid), (2)Basic (have positively charged side chains, like lysine, arginine, and histidine), (3)Polar uncharged (have side chains that can form hydrogen bonds, like serine and threonine), and (4)Nonpolar (have hydrophobic side chains, like leucine, valine, and phenylalanine). These classifications help predict how amino acids behave in water, how they interact within a protein, and how proteins fold into their final functional shapes.

• D and L-Amino Acids:L-form predominates in biological systems because enzymes that synthesize and process proteins are specific to this configuration.

• Peptide Bonds:This bond forms between the carboxyl group of one amino acid and the amino group of another, releasing a molecule of water.

• The 20 Natural Amino Acids: Categorized based on their side-chain properties into acidic, basic, polar, and nonpolar groups.

Acidic Amino Acids

Acidic amino acids are a unique group of amino acids that contain extra carboxyl (-COOH) groups in their side chains. These additional carboxyl groups can donate protons (H⁺) when in a solution, especially at physiological pH (~7.4), which causes them to become negatively charged. This negative charge is important because it affects how these amino acids interact with other molecules. Their ability to release a proton and carry a negative charge makes them excellent participants in ionic (electrostatic) interactions—a critical feature in maintaining protein structure and function, especially when interacting with positively charged groups like metal ions or basic amino acids.

Two key acidic amino acids are aspartic acid (abbreviated as Asp, one-letter code D) and glutamic acid (Glu, one-letter code E). Aspartic acid has a shorter side chain (just one carbon between the alpha carbon and the carboxyl group), which makes it more compact and often places it in enzyme active sites, where it can directly participate in catalysis or substrate binding. Because of its negative charge, aspartic acid is often involved in forming salt bridges—strong ionic bonds with positively charged residues like lysine or arginine—which help stabilize protein structure or enzyme-substrate complexes.

Glutamic acid, on the other hand, has a longer side chain (two carbons before the terminal carboxyl group), giving it slightly more flexibility. It plays a prominent role in cell signaling and metabolism, especially in the nervous system, where it acts as a key excitatory neurotransmitter. In proteins, glutamic acid contributes to protein folding and binding by interacting with positive charges or forming hydrogen bonds. It also plays a role in protein-protein interactions, helping stabilize complexes or guiding enzymes to their targets.

In summary, both aspartic acid and glutamic acid are essential for the structural integrity and function of proteins due to their negative charges, ability to donate protons, and involvement in electrostatic interactions, enzyme function, and signaling pathways.

Both acidic amino acids contain carboxyl (-COOH) side chains, which donate protons at physiological pH, making them negatively charged.

• Aspartic Acid (Asp, D): Often found in enzyme active sites, where it participates in acid-base catalysis and forms ionic interactions.

• Glutamic Acid (Glu, E): Plays a role in protein structure and interactions via ionic bonding; its free form (glutamate) functions as a key neurotransmitter in the brain.

Basic Amino Acids

Basic amino acids are characterized by their side chains that contain nitrogen-rich functional groups capable of accepting protons, making them positively charged at or near physiological pH (around 7.4). This positive charge plays a crucial role in how these amino acids interact with negatively charged molecules such as DNA, phosphate groups, or acidic amino acids. The three main basic amino acids are lysine, arginine, and histidine, and each has a unique chemical structure and specific biological functions.

Lysine (Lys, K) has a long, flexible aliphatic side chain ending in a primary amine group (-NH₃⁺ at physiological pH), which is highly basic and remains positively charged in most biological environments. This allows lysine to form strong ionic interactions and hydrogen bonds, making it vital in protein-protein interactions, particularly in protein complexes and structural motifs. Additionally, lysine residues are frequent targets of post-translational modifications, especially acetylation, methylation, and ubiquitination, which play a central role in regulating gene expression. For instance, in histones, which help package DNA, lysine residues on their tails are often acetylated to loosen DNA-histone binding, allowing genes to become more accessible for transcription.

Arginine (Arg, R) has a side chain containing a guanidinium group, which is a planar, highly basic group that can form multiple hydrogen bonds and strong electrostatic interactions. This makes arginine especially useful in binding negatively charged molecules like DNA, RNA, and phosphate groups. It is also essential in metabolic pathways such as the urea cycle, where it helps eliminate excess nitrogen from the body. Additionally, arginine is the precursor for nitric oxide (NO), a critical signaling molecule involved in vasodilation, immune responses, and neurotransmission. The ability of the guanidinium group to stabilize multiple interactions makes arginine-rich regions crucial for binding specificity and regulation.

Histidine (His, H) is unique among the basic amino acids because of its imidazole side chain, which has a pKa close to physiological pH (~6.0–6.5). This means that histidine can switch between a protonated (positively charged) and unprotonated (neutral) state under small pH changes. As a result, histidine acts as a versatile proton donor or acceptor, making it an essential residue in enzyme active sites where proton transfer is needed during catalysis. This property is why histidine is frequently involved in acid-base catalysis and in stabilizing reaction intermediates.

One of histidine's most important applications in molecular biology is in protein purification using Ni-NTA (Nickel-Nitrilotriacetic Acid) affinity chromatography. This technique exploits the fact that the imidazole ring of histidine has lone pair electrons that can coordinate with nickel ions (Ni²⁺). In this method, proteins are engineered with a histidine tag (His-tag)—usually a string of six or more histidines (6x-His tag)—on either the N- or C-terminus. When the tagged protein mixture is passed through a Ni-NTA resin, the histidine residues bind tightly to the nickel ions immobilized on the column, allowing the tagged protein to stick while other proteins wash through. The protein of interest is then eluted by adding imidazole to the buffer, which competes with the histidine residues and displaces them from the nickel. This method is widely used because it’s specific, efficient, and gentle, preserving protein structure and function.

In summary, lysine, arginine, and histidine are essential basic amino acids that support protein structure, biochemical reactions, and molecular interactions. Histidine’s imidazole group is especially versatile, not only in catalysis but also in biotechnology through its critical role in Ni-NTA purification.

These amino acids have side chains that accept protons, making them positively charged at physiological pH.

• Lysine (Lys, K): Important for protein-protein interactions and histone modifications.

• Arginine (Arg, R): Participates in the urea cycle and nitric oxide synthesis.

• Histidine (His, H): Has an imidazole group that acts as a proton donor/acceptor in enzymatic reactions.

Polar Amino Acids

Polar amino acids have side chains that contain electronegative atoms—such as oxygen or nitrogen—which make them hydrophilic (water-loving) and capable of forming hydrogen bonds with water and other polar molecules. These interactions are essential for protein solubility, protein folding, and biochemical reactions, especially in aqueous environments like the cytoplasm or extracellular fluid. Unlike acidic or basic amino acids, polar amino acids do not carry a net charge at physiological pH, but their side chains can participate in bonding that stabilizes protein structures and supports molecular recognition.

Two important polar amino acids are serine (Ser, S) and threonine (Thr, T). Both of these amino acids have hydroxyl (-OH) groups in their side chains. These -OH groups are highly reactive and play a critical role in hydrogen bonding and post-translational modifications, particularly phosphorylation. In phosphorylation, a phosphate group (PO₄³⁻) is enzymatically added to the hydroxyl group, often regulating protein activity, signaling pathways, and protein-protein interactions. For example, serine and threonine residues in enzymes or receptors are often phosphorylated to activate or deactivate their function, making them key players in cell signaling cascades such as those driven by kinases (like in insulin signaling or MAPK pathways).

Asparagine (Asn, N) and glutamine (Gln, Q) are also polar but differ because their side chains contain amide groups (-CONH₂) instead of hydroxyls. These amide groups are polar but uncharged, and they are excellent at forming hydrogen bonds, making Asn and Gln crucial for maintaining protein stability and hydration. They are often found on the surface of proteins, where they can interact with water, or in the core of protein structures, where they participate in hydrogen bonding networks that stabilize secondary and tertiary structure. They are also involved in molecular recognition and protein-DNA or protein-RNA interactions, thanks to their ability to form specific and directional hydrogen bonds.

To help students easily remember the structures of asparagine (Asn) and glutamine (Gln), it helps to compare them to their acidic counterparts, aspartic acid (Asp, D) and glutamic acid (Glu, E). Asp and Glu both have carboxylic acid (-COOH) groups in their side chains, which can lose a proton and become negatively charged. Asn and Gln are structurally similar—but instead of a carboxylic acid group, they have an amide group (-CONH₂) in the same position. In other words, if you "convert" the -OH part of the carboxyl group into an -NH₂ group, you go from Asp to Asn or from Glu to Gln. This structural similarity makes them easy to pair and memorize:

• Asp (D) → Asn (N) = 1-carbon chain ending in -COOH vs. -CONH₂

• Glu (E) → Gln (Q) = 2-carbon chain ending in -COOH vs. -CONH₂

Polar amino acids contain side chains with electronegative atoms like oxygen or nitrogen, enabling them to form hydrogen bonds with water, other amino acids, or biomolecules, which is essential for protein structure and function.

• Serine (Ser, S) & Threonine (Thr, T):Contain hydroxyl (-OH) groups that can form hydrogen bonds and serve as common sites for phosphorylation, playing key roles in cell signaling and regulation.

• Asparagine (Asn, N) & Glutamine (Gln, Q): Contain amide side chains that enable them to participate in hydrogen bonding, stabilizing protein structures and mediating molecular interactions.

Nonpolar Amino Acids

Nonpolar (hydrophobic) amino acids have side chains that lack electronegative atoms and are composed mainly of hydrocarbon groups—making them water-insoluble and nonpolar. These residues are crucial in protein folding, because they tend to cluster together away from water in the interior of proteins, creating a hydrophobic core. This clustering helps proteins achieve their proper three-dimensional shape and stability. They also play a major role in membrane-associated proteins, where nonpolar side chains interact with the lipid bilayer's hydrophobic tails.

Let’s start with glycine (Gly, G), the smallest of all amino acids. Unlike other amino acids that have a distinct R group, glycine's side chain is simply a hydrogen atom. Because of this, the alpha carbon in glycine is not chiral, and the amino acid is extremely flexible. It can fit into tight spaces within protein structures—especially in turns, loops, and bends of polypeptide chains. For example, glycine is often found in beta-turns and collagen triple helices, where steric hindrance would prevent larger side chains from fitting. This makes glycine essential for regions of proteins that need to twist or fold sharply, adding flexibility and mobility to the overall structure. In protein folding, glycine is like the swivel joint that allows proteins to bend or make tight turns. For example, when a protein needs to form a sharp U-turn (like in a beta turn), glycine is often there to make that turn smooth and sharp without creating a clash.For example, in collagen, one of the most abundant proteins in your body, every third amino acid is glycine. That’s because collagen forms a triple helix, and without glycine’s small size and flexibility, the helix would be too bulky and unstable.

Next, tyrosine (Tyr, Y) and tryptophan (Trp, W) are often confusing because they contain polar functional groups—a hydroxyl (-OH) group on tyrosine and an indole nitrogen (-NH) group on tryptophan. However, these amino acids are still considered predominantly nonpolar because the bulk of their side chains is made up of large aromatic (ring) systems. These rings are hydrophobic and dominate the molecule’s behavior in aqueous environments. Their polar groups can form hydrogen bonds, but the electron-rich aromatic rings favor interactions with other hydrophobic or aromatic regions in proteins, like in pi-pi stacking. This duality makes Tyr and Trp important in enzyme active sites and membrane proteins, where both hydrophobic packing and limited hydrogen bonding are needed. Overall, these two amino acids are aromatic, meaning they have large ring structures in their side chains. These rings are made mostly of carbon and hydrogen, which makes them nonpolar and hydrophobic overall. However, they each contain a small polar group that can still form hydrogen bonds that are important for many biological functions. For example,Tryptophan is involved in the synthesis of serotonin, a neurotransmitter. Its indole ring is important for passing through cell membranes, thanks to its hydrophobic character. Also, in proteins embedded in membranes, like G-protein-coupled receptors (GPCRs), you often find tyrosine and tryptophan at the membrane-water boundary—they anchor the protein and interact with both the hydrophobic membrane interior and the aqueous exterior.

Methionine (Met, M) is another nonpolar amino acid that contains a sulfur atom in its side chain. This sulfur is part of a thioether group, where the sulfur is bonded to a methyl group (-CH₃). The methylation of sulfur masks its polarity, making methionine’s side chain nonpolar and hydrophobic, despite the presence of a sulfur atom. This allows methionine to participate in hydrophobic interactions rather than forming hydrogen bonds or reacting with polar molecules. Again, Methionine has a thioether group in its side chain, which means it contains a sulfur atom bonded to a methyl group (–CH₃). Normally, sulfur might add polarity, but in methionine, the methyl group “shields” the sulfur, making the entire side chain nonpolar.

An important feature of methionine is that it is the first amino acid incorporated into proteins during translation in nearly all organisms. In eukaryotes and archaea, this is typically initiator methionine (Met), while in prokaryotes, it is N-formylmethionine (fMet). The reason for this is evolutionary and functional. The AUG codon, which codes for methionine, serves as the universal start codon in the genetic code. Translation machinery (ribosomes and initiation factors) has evolved to recognize AUG as the signal to start translation. Over time, methionine became locked into this role, likely because it is chemically stable, relatively neutral (nonpolar but not too bulky), and does not interfere with the early folding of the nascent polypeptide. After translation, methionine is often cleaved off by enzymes if it's not needed in the final protein structure. Thus, its role as a starter is more functional than structural, driven by the evolution of translation systems rather than its chemical properties alone.So methionine is like the “green light” that tells the ribosome to start building a protein, and it doesn’t cause drama—it gets things going and then sometimes even steps aside (many proteins have their initial methionine cleaved off after translation).

In summary, nonpolar amino acids like glycine, tyrosine, tryptophan, and methionine contribute to protein shape, stability, and localization in unique ways—from adding flexibility to forming hydrophobic cores to initiating translation. Understanding their properties helps explain how proteins fold and function in complex biological systems.

These amino acids have side chains that are largely nonpolar, contributing to hydrophobic interactions that drive protein folding, stability, and membrane association.

• Glycine (Gly, G): The smallest amino acid, glycine contributes flexibility to protein structures and is often found in tight turns or loop regions due to its minimal steric hindrance.

• Tyrosine (Tyr, Y) & Tryptophan (Trp, W): Tyrosine and tryptophan have large aromatic side chains that contribute to hydrophobic interactions. Although predominantly nonpolar,their hydroxyl (-OH) and indole nitrogen (-NH) groups allow them to also engage in hydrogen bonding, making them amphipathic.

• Methionine (Met, M): Methionine contains a nonpolar thioether side chain (-S-CH₃) that contributes to hydrophobic interactions, particularly in the protein interior.

Nomenclature of Peptides

Amino acids are the fundamental building blocks of peptides and proteins, each of which is structured around a central carbon, called the alpha (α) carbon, which is bonded to four different groups: a hydrogen atom, an amino group (-NH₂), a carboxyl group (-COOH), and a distinctive R group (or side chain). The R group is what differentiates one amino acid from another and defines its chemical properties (such as being nonpolar, polar, acidic, or basic). When amino acids are linked together in a chain, they form peptides, with the N-terminus (the amino group side) on one end and the C-terminus (the carboxyl group side) on the other. This specific orientation is critical because it determines the peptide's structure and function.

The process of peptide bond formation begins when the amino group of one amino acid reacts with the carboxyl group of another, releasing a molecule of water (a condensation reaction). The bond that forms between the two amino acids is called a peptide bond, and the result is a dipeptide (two amino acids linked together). As more amino acids join the chain through similar peptide bonds, the structure grows into a polypeptide. The directionality of the polypeptide chain is always from the N-terminus to the C-terminus, meaning that proteins are always synthesized in this order by ribosomes during protein translation.

In terms of drawing amino acids, the N-terminus is usually drawn on the left, and the C-terminus is drawn on the right. Each amino acid's structure includes the central carbon (α-carbon) attached to:

• The amino group (-NH₂) on the left side (N-terminus).

• The carboxyl group (-COOH) on the right side (C-terminus).

• The hydrogen atom and the R group (side chain) are attached to the α-carbon.

When drawing a peptide, you would typically draw the backbone of the chain (the repeating sequence of N-C-C between each amino acid) and then show the side chain (R group) sticking out from each α-carbon. The R group plays an important role in the final shape of the protein because it dictates how the peptide interacts with other molecules. As the chain folds into its three-dimensional shape, the R groups flip back and forth, orienting themselves in ways that minimize or maximize interactions with other molecules, especially water. Hydrophobic side chains tend to hide inside the protein’s core, away from water, while hydrophilic side chains may be exposed on the outside to interact with water or other molecules.

The sequence of amino acids in a peptide is critical because it dictates the secondary, tertiary, and even quaternary structures of the protein, which ultimately determine its biological function. The N-terminus to C-terminus directionality is not just an arbitrary naming convention but reflects the sequence in which the amino acids are coded by DNA and then assembled by the ribosome during translation. For example, if a protein’s amino acid sequence is Serine-Glycine-Leucine, the serine would be at the N-terminus and the leucine would be at the C-terminus.

The process of protein folding occurs after the peptide chain is synthesized. As the chain folds, the R groups of the amino acids influence how the chain bends, twists, and forms secondary structures (such as alpha helices or beta sheets). These secondary structures are stabilized by interactions between the side chains, such as hydrogen bonds, ionic bonds, or hydrophobic interactions. As the protein folds into its tertiary structure, the R groups continue to play a critical role, driving the protein toward its functional, three-dimensional shape. For example, in a protein like hemoglobin, the specific sequence of amino acids from the N-terminus to the C-terminus determines how the protein folds and how it can bind to oxygen. Mutations in this sequence can cause the protein to fold incorrectly, as seen in sickle cell disease, where a single amino acid change in the sequence causes hemoglobin to form an abnormal shape, leading to health problems.

1. Naming Peptides: The N-terminus (amino group) is always at the start of the peptide chain, and the C-terminus (carboxyl group) is at the end. This directionality is fundamental because it reflects the sequence in which amino acids are added during protein synthesis.

2. Drawing Amino Acids: The general structure of each amino acid is N-terminus – α-carbon – C-terminus, with the R group attached to the α-carbon. When drawing a peptide, you represent the backbone of the chain (N-C-C) and show the R groups attached to the α-carbons.

3. The Role of R Groups: The R groups flip back and forth as the polypeptide folds into its final three-dimensional shape, affecting how the protein interacts with other molecules, its stability, and its function.

4. Function and Sequence: The sequence of amino acids in a polypeptide (from N-terminus to C-terminus) determines the folding and function of the protein, with small changes in the amino acid sequence potentially leading to significant biological consequences.

By understanding these concepts and being able to draw and name amino acids and peptides, students will gain a strong foundation in how proteins are built, how their structures determine their function, and how small changes in the amino acid sequence can lead to profound changes in protein behavior and cellular processes.