The endless diversity created by just 20 amino acids
The structure and function of a protein depend on how its amino acids are arranged. Before diving into this, let's first get a feel for the incredible diversity that proteins can have. There are about 20 standard amino acids commonly used in living things. Now, imagine using these 20 amino acids to make a very, very short peptide consisting of just 10 amino acids. The first position can take any of the 20 amino acids, and the second position can also take any of the 20. If each of the 10 positions independently has 20 possibilities in this way, the number of different amino acid sequences you can make for the peptide is 20¹⁰, which is about 10¹³, or over 10 trillion. Of course, not all of these sequences actually exist as real proteins or form stable structures. The proteins used in actual living organisms are only a tiny fraction of the countless sequences theoretically possible. Even so, the fact that we can make such a massive number of different sequences using just about 20 amino acids really shows us where protein diversity comes from.
In reality, actual proteins are usually made up of much more than 10 amino acids. The longer the sequence gets, the faster the number of possible combinations grows beyond our imagination. In other words, as the saying goes, numbers have power—I want to emphasize that this incredible possibility of combinations serves as an important foundation that allows proteins to create countless diverse structures inside living organisms and perform different functions. Showing you these numbers is a nice little warm-up to help explain the importance of proteins.
Carbohydrates, lipids, nucleic acids, and proteins are listed as the essential biological macromolecules for life. Among these, proteins are by far the most abundant and perform the most diverse functions. If you were to completely dry out a cell—which is a watery environment—by squeezing out all the water, probably most of the remaining components would be proteins. Proteins not only form the cellular skeleton to provide structural support, but they also move substances in and out of cells and perform countless biochemical functions inside cells. Thousands of enzymes that speed up chemical reactions, transporters and ion channels that move substances across cell membranes, and receptors that receive, interpret, pass along, and amplify signals from outside the cell are all proteins. Signaling hormones, antibodies for immune defense, and hemoglobin that carries oxygen and nutrients are also proteins. Proteins are truly the cellular workers that directly carry out almost all functions and activities of a living organism. Therefore, even cells with the same genetic information can have different identities and functions depending on which proteins they produce. Usually protein is introduced as one of the three major nutrients along with carbohydrates and fats, but its role and meaning are far too overwhelmingly important to simply think of it as just a nutrient. Then, how can proteins perform such diverse and amazing roles? To understand this, let's first look at the very basics of proteins.
The basic building blocks that make up proteins in living things are about 20 kinds of amino acids, and each has its own unique side chain (R-group). These side chains have different chemical properties such as polarity, charge, and size. Depending on how amino acids are combined and arranged, and how their side chains interact with one another, proteins can form a wide variety of three-dimensional structures. Structural diversity is very important for proteins because this diversity is directly linked to functional diversity. That is why people often say, protein structure determines function.
Protein Structure (From Primary to Quaternary)
Let's look at how proteins form various shapes. Protein structure is generally divided into four levels, ranging from the most basic amino acid sequence to complex structures where multiple polypeptide chains combine. First, when amino acids are connected long together through peptide bonds, a single polypeptide chain is formed. The specific order in which these amino acids are connected—that is, the arrangement and sequence of amino acids—is called the primary structure. This amino acid sequence becomes the absolute foundation that determines what kind of structure the protein can form later. After the polypeptide chain is made, a certain shape appears in some parts of the chain. Hydrogen bonds form between atoms in the polypeptide backbone, creating a regular and repetitive folding pattern. This local folding is called the secondary structure, and as various interactions are added in multiple places along with this secondary structure, the entire polypeptide chain folds into a unique three-dimensional shape. This overall three-dimensional shape created by a single polypeptide chain is called the tertiary structure. Finally, multiple polypeptide chains, each having its own tertiary structure, sometimes bind together to form a single functional protein complex. This is called the quaternary structure.
Hydrogen Bonds Between Backbones: α-helix and β-strand
Let's take a closer look at these structures. First, the primary structure of a protein refers to the order of amino acids—meaning the sequence—created when amino acids are connected by peptide bonds. A dehydration reaction occurs between the carboxyl group (-COOH) of one amino acid and the amino group (-NH₂) of another, forming a peptide bond between the two. As this bond repeats, a long polypeptide chain is created. In this polypeptide backbone, bonds between the carbonyl group (C=O) and the amide N-H exist repeatedly. The oxygen of the carbonyl group has a partial negative charge (δ⁻), while the hydrogen of the N-H bond has a partial positive charge (δ⁺), so when the two are located at the right distance and direction, they can form a hydrogen bond. And it is precisely these repetitive hydrogen bonds between backbones that stabilize the polypeptide chain into a certain shape, creating secondary structures like the α-helix and β-strand.
First, how is the α-helix formed? Within the same polypeptide chain, if the carbonyl oxygen (C=O) of one amino acid residue regularly repeats a pattern of hydrogen bonding with the amide hydrogen (N-H) of the amino acid residue 4 positions down the line, the chain gradually curls in one direction to take on a spiral shape. It's a principle similar to how a spiral staircase is made by slightly shifting the direction of each step. On the other hand, if the polypeptide chain takes an extended shape and hydrogen bonds occur between the backbones of nearby parallel chain sections, a β-strand is created. When these strands are arranged side by side and additionally form hydrogen bonds with each other, a pleated sheet-like β-sheet structure is formed. The α-helix and β-strand formed this way are connected to each other by relatively flexible regions called loops or turns. And as these secondary structures and interactions between various side chains work together, the entire polypeptide chain folds into a specific three-dimensional shape, which is the tertiary structure of the protein.
Stabilization of Tertiary Structure: Chemical Properties of Side Chains
The tertiary structure is further stabilized not only by hydrogen bonds between backbones, but also through hydrophobic interactions between side chains, hydrogen bonds, ionic interactions, and sometimes disulfide bonds. Chemical properties such as polarity, hydrophobicity, and charge possessed by these side chains affect the overall structure of the protein. For example, water-fearing hydrophobic residues tend to gather together inside the protein structure to avoid contact with water, whereas highly polar or charged residues are often positioned on the outside (surface) of the structure to contact the surrounding watery environment. As these interactions achieve an overall balance, the protein forms its structure in a direction that lowers free energy in a given environment. Finally, when different polypeptide chains, each forming an independent three-dimensional structure, combine to form a single functional protein complex, the spatial arrangement between these chains is called the quaternary structure, and each polypeptide chain is called its subunit.
Conformational Change of Proteins
The reason protein structure is so important is not simply because proteins need to have a specific shape. It is because proteins change their structures as needed and use these structural changes themselves to regulate their functions. A protein is not a molecule that exists in one completely fixed form. Proteins can move back and forth between multiple structural states while constantly interacting with their surrounding environment, and when a specific structural state among them is formed, they can bind to substrates or interact with other proteins to promote chemical reactions.
When a ligand binds to a receptor, the acidity (pH) or ion concentration changes, or chemical changes such as phosphorylation occur, interactions inside the protein can change. As a result, the overall shape of the protein or the structure of a specific region changes, allowing it to perform functions it couldn't perform before, or conversely, stopping it from performing its existing functions anymore. This change is called a conformational change.
This phenomenon is easy to understand if we look at the active site of an enzyme. Enzymes are giant proteins mostly made by one or more polypeptide chains, and inside them is an active site that binds to specific molecules and causes chemical reactions. The active site is a sophisticated space created by specific residues gathering close together. Specific residues must be arranged at the exact distance, direction, and correct charge state to bind with a substrate. In some cases, the active site is pre-formed in a relatively stable shape, but for most enzymes, as a substrate approaches or binds, a conformational change can occur around the active site, which helps the enzyme and substrate fit together even better.
Earlier, we mentioned that the secondary and tertiary structures of proteins are maintained by hydrogen bonds between backbones, as well as hydrophobic interactions, hydrogen bonds, ionic interactions, and disulfide bonds between amino acid side chains. Saying that a protein undergoes a conformational change does not mean that all these various bonds completely break and get remade entirely from scratch; rather, it is a process where the relative positions of atoms and residues inside the protein are subtly rearranged. Some interactions may become weaker, and new interactions may form, so two parts that were close to each other might move apart, or conversely, parts that were far away might come close together. When these small changes add up, the shape and chemical environment of the active site change. Consequently, the active site becomes in a state well-suited to bind with a specific substrate, helping the chemical reaction occur smoothly while fixing the substrate in the exact position. In the active site, there are generally residues that bind the substrate in the proper position, and catalytic residues directly involved in chemical reactions such as electron transfer or the formation or cleavage of covalent bonds. A protein's function is not determined solely by what structure it has. Proteins can change their structures to turn their functions on and off, change how they bind to other molecules, and switch to new functional states according to cell signals. Now let's look at a few specific examples of how these structural changes actually happen in living environments.
Digestive Enzymes and Acidity (pH)
When we eat food, pepsin, a powerful protein-digesting enzyme secreted in the stomach, digests large chunks of steak while (fortunately) protecting the stomach lining without digesting it. The main reason for this is that pepsin is a zymogen enzyme, which turns into an active enzyme only when specific environments or conditions are met. Pepsin is not made in an active state from the beginning; it is secreted in an inactive form called pepsinogen, and only when enough stomach acid is secreted to make the inside of the stomach a highly acidic environment with a very low pH does it undergo a structural change and convert from pepsinogen into the active enzyme pepsin. In other words, this is an example where pH changes charges to induce conformational change.
Aspartic acid (Asp) and glutamic acid (Glu), which make up the active site of the pepsinogen enzyme, belong to acidic amino acids because they have an additional carboxylic acid group (-COOH) on their side chains in addition to the backbone. Normally, in a physiological neutral state, they lose a proton (H⁺) and carry a negative charge as -COO⁻, but if the environment changes to a highly acidic one and protons become quite abundant around them, the rate of accepting them increases, shifting the equilibrium toward becoming -COOH, thus decreasing the negative charge. This change in charge affects the electrostatic interactions and hydrogen bonds inside the protein, and as a result, the structure of pepsinogen changes, cutting away the part that usually covers the enzyme's active site and activating it into pepsin. This process is not just about enzymes working better in an acidic environment; it is a representative example where a change in pH alters the charge state of amino acids, and that change alters the protein's structure, leading to enzyme activation.
Calmodulin and Calcium Ions
Now let's look at calmodulin, a calcium ion (Ca²⁺) sensor protein, as a representative example of how ions cause conformational changes in proteins. Calcium ions act as important second messengers in intracellular signaling systems. Cells normally maintain a very low concentration of Ca²⁺ in the cytoplasm, but when they receive external stimuli, the Ca²⁺ concentration in the cytoplasm can temporarily increase. Cells use this concentration change as an important signal, and the core protein that connects this change in Ca²⁺ concentration to actual biochemical reactions is calmodulin.
Calmodulin itself is not an enzyme. Instead, it acts as a regulatory protein that receives Ca²⁺ signals and converts them into activity changes in various enzymes and proteins. Calmodulin has four Ca²⁺-binding sites, which consist of structures rich in negatively charged aspartic acid and glutamic acid residues. When Ca²⁺ binds to these sites, the structure of calmodulin changes significantly. As a result, the hydrophobic binding surface that was usually hidden inside the molecule is exposed to the outside, putting this hydrophobic surface in a state where it can selectively bind to specific target proteins. When calmodulin binds to a target protein, that protein also undergoes a conformational change, which can activate or inhibit the enzyme. In other words, calmodulin is not just a protein that catches calcium; it is a molecular sensor that reads changes in calcium ion concentration as changes in protein structure, and then transmits those structural changes into functional changes in other proteins. Activated calmodulin targets a wide variety of enzymes and proteins, and is broadly involved in various cell functions essential for sustaining life, such as muscle contraction, apoptosis, nerve cell signaling and memory formation, and gene expression regulation.
Kinases and Magnesium Ions (Mg²⁺)
Another outstanding case where a protein's function is regulated by a specific ion is a kinase. Kinases are very important regulatory enzymes inside cells, usually playing the role of taking a phosphate group off ATP and transferring it to another protein or molecule. This phosphorylation can change the protein's structure and function, acting somewhat like an ON/OFF switch inside the cell. However, phosphorylation has a problem. Under physiological conditions, the three phosphate groups making up ATP each carry negative charges, making it an unstable molecule that already experiences internal repulsion within itself. Furthermore, as we saw earlier, the Asp and Glu amino acids in the kinase's active site also generally have strong negative charges, so electrical repulsion pushes like-charges away from each other between them. Therefore, in order to bind ATP at the exact position in the kinase's active site and allow the phosphate group transfer reaction to happen, this negative charge must be regulated. And the magnesium ion (Mg²⁺) plays an important role here.
Mg²⁺ first binds to ATP, partially covering the charge around the phosphate group to be detached, and helps ATP maintain the proper position and orientation in the kinase's active site. As a result, the reaction transferring the phosphate group to the substrate can happen much more easily and smoothly. Rarely but sometimes in some enzymes, manganese ions (Mn²⁺) play that role instead of Mg²⁺. Of course, this is different from cases like the earlier calmodulin where the binding of Ca²⁺ changes the overall shape of the protein, but the role of Mg²⁺ in kinases acts as an important factor helping chemical reactions occur by regulating the charge environment of the active site and the placement of ATP. We can see that ions affect proteins in various ways.
How Do Proteins Regulate This Complex Living System?
Now let's think from a slightly bigger perspective. As we looked at earlier, protein structure is not fixed. Protein structures can change depending on various factors such as pH changes, ion binding, interactions with other molecules, and chemical modifications. And this conformational change does not simply change the shape of the protein. When the structure changes, the way it binds to other molecules changes, the shape of the active site changes, and ultimately the function of the protein itself can change. The activation of pepsinogen, the calcium binding of calmodulin, and the interaction of kinases with Mg²⁺ that we looked at earlier are all examples showing this fact. In other words, proteins are not just molecules that exist inside cells and repeat set tasks. They are highly dynamic molecules that sense various signals coming from inside and outside the cell, and regulate cell activity by changing their own structure and function according to those signals. And among these proteins, the most representative protein is the enzyme.
Enzymes do not just play the role of making chemical reactions happen quickly. Inside cells, they play a role in delicately regulating the overall activity of the cell by influencing what chemical reactions can happen, under what conditions, and how fast. Thousands of enzymes and various other proteins exist in living cells. Enzymes do not work independently of each other; they are connected to one another. The product made by one enzyme becomes the substrate for the next enzyme, and multiple reactions connect and continue to form a complex metabolic network. Therefore, even a slight change in the activity of a single enzyme can alter the next step of the reaction and the flow of the entire pathway. That is why, for cells to operate stably in a constantly changing environment, the reactions of these countless enzymes and the activities of proteins must be regulated delicately. In other words, this is why it is important which enzyme should work, and when and where it should work, how much of it should exist, or how fast it should react.
Then a question arises here. Do cells always make and use all the necessary proteins in advance in sufficient quantities? Of course not. Because making proteins requires a considerable amount of energy and materials. If cells kept making and storing all proteins to their maximum amount in advance just because there is a possibility they might use them, it would be a huge waste of resources and a very inefficient and heavy burden from the cell's perspective. So instead, cells use a much more efficient strategy. That is, they produce the necessary proteins only at the needed time, in the needed place, and in the needed amount. Instead of pre-storing all proteins in a finished state, cells store the genetic information needed to make proteins in the form of DNA. When a specific protein becomes necessary, the cell selectively reads the related genetic information and produces the required protein based on that information. All cells basically store the same DNA inside the nucleus, but depending on the cell type, function, and situation it faces, which genetic information is taken out and read and what proteins are produced differ from one another.
What is more important is that DNA is not just a simple information storage room. In response to external environments and internal signals, cells regulate which genes to read, in which cells to read them, and how much to read them. And countless proteins, including transcription factors and signaling proteins, feed back into this very regulatory process. Interestingly, it creates a feedback loop here. DNA stores the information to make proteins, but proteins are the very agents that regulate when and how to use the information in DNA. In other words, proteins not only perform the actual work of the cell, but also manage and regulate the activities of other proteins, transmit cell signals, and, when necessary, read the information stored in DNA to regulate the production of new proteins. Ultimately, if we separate DNA and proteins and try to understand them individually, we cannot truly understand life. To understand this interdependent relationship between DNA and proteins—where DNA holds the blueprint for proteins, and proteins control the expression of DNA—we are now going to begin a very detailed and rich story about DNA, the molecule that stores and transmits the genetic information of living things.
