2.3: Structure and Function of Membrane Proteins

Learning Goals

• Explain how the primary sequence and the environment of a protein influence its final 3D structure, specifically with respect to the different types of intermolecular forces that it will form with itself and its environment.
• Distinguish between integral and peripheral proteins with regard to their solubility properties, structure, and manner of attachment to membranes.
• Describe the different roles of membrane proteins within a biological membrane.
• Describe the structure and use of plasma membrane–adjacent structures like the animal cell extracellular matrix and the plant cell wall.

Earlier in this chapter, we learned that a biological membrane is different from a phospholipid bilayer. A biological membrane also has proteins embedded in it that will change its properties and add functions. In this topic, we will first review the formation of proteins from amino acids, then discuss the characteristics of membrane proteins. We will also examine the unique properties of the plasma membrane as the barrier that separates the cellular contents from the outside world. We end with a brief discussion of what’s on the outside of a cell—namely, the extracellular matrix (animals) or a cell wall (plants, algae, and fungi).

The protein content of a membrane can range from ~25% (in the case of the myelin sheath cells of neurons) to nearly 80% (for the mitochondrial inner membrane), with more typical membranes consisting of protein and lipids in an approximately equal ratio (50:50) by weight.

Because of the associated proteins, the thickness of biological membranes is almost always thicker than that of a simple lipid bilayer. Biological membranes are typically 6.5 to 10 nm thick. A lipid bilayer without proteins is about 5.5 nm thick.

Brief Review of Amino Acids and the Chemistry of Protein Folding

We assume you have preexisting knowledge about amino acids and proteins, so we are approaching this as a review. As always, we encourage you to explore the resources in the introduction if you need a refresher.

There are 20 different types of amino acids that are used to form proteins. Each amino acid contains an amino group (NH₃⁺) and a carboxylic acid group (COO^–). In between is a carbon atom connected to a variable chemical structure called the R group or side chain. To make a protein, the amino group from one amino acid will covalently bond with the carboxylic acid group of another amino acid to create a peptide bond. This creates a repeating N–C–C pattern when the amino acids are strung together. This repeating N–C–C is called the protein backbone.

The sequence of the amino acids in a polypeptide is based on the sequence of codons that are read from the mRNA by the ribosome during translation. We call the order of the amino acids in the polypeptide chain the primary structure or primary sequence.

There are many, many versions of side chain charts on the internet for you to find, each designed with a slightly different learner in mind, which is why we have not included one here.

The R groups are what give each amino acid its distinct chemical properties. Some R groups are large, and some are small; some are negatively charged at physiological pH, and some are positively charged. It’s worth taking some time to look at the chemical structure of the R groups and start investigating how these chemical groups impact protein structure/function.

The order of the amino acids in the primary structure plays a crucial role in determining folding and function of a protein. A given protein will fold in a very specific way depending on the molecular interactions of the amino acids in its primary structure. A variety of intermolecular forces (i.e., hydrogen bonds, ionic bonds, and induced dipole–induced dipole/van der Waal forces) contribute to the final 3D fold of the protein. Proteins will, many times, spontaneously fold into the shape that is the most stable and requires the least amount of energy to maintain. Other times, chaperone proteins will help facilitate the proper folding of a protein. We will learn about chaperones in more detail in Chapter 4.

When discussing protein folding, we split the different interactions into four categories, which we call the “levels” of protein folding. It’s important to remember that these do not happen sequentially. Instead, they happen more or less all at once in different parts of the protein as it folds into its final 3D shape. These levels are as follows:

• Primary structure forms when amino acids are covalently bound together by peptide bonds. The order of the amino acids is the key feature of the primary structure.
• Secondary structure forms when the backbone interacts with itself via hydrogen bonds. It forms a repeating local pattern, commonly with nearby amino acids. Examples of this structure include alpha helices and beta sheets.
• Tertiary structure forms when R groups get involved. They interact with either other R groups or the backbone, usually via intermolecular forces like the ones mentioned above.
• Quaternary structure forms when different polypeptide chains come together to form a protein (or protein complex). All of the same intermolecular forces get used as we would expect in tertiary structure.

Disulfide bridges are the only other covalent bond used in folding aside from the peptide bond that creates the primary structure. Disulfide bridges form between two cysteine residues. They can play a role in tertiary or quaternary structure but are far less common than the other bonds. To compare, there are usually only a handful of disulfide bridges in a protein, if any, whereas there may be hundreds, or even thousands, of hydrogen bonds. As a result, we tend to discuss disulfide bridges much less often, but that doesn’t mean that they should be forgotten.

The chemical environment (especially pH) also plays a very important role in protein folding. A soluble protein in the cytosol will experience different intermolecular forces than a protein that is embedded in a membrane. In each case, the protein will respond to its folding environment. Unlike the soluble proteins of the cytosol, many membrane proteins must be able to embed themselves in the lipid bilayer, including the nonpolar tail region. This requirement impacts how the protein folds and which amino acids of its primary structure are expected to be on the exterior in each region.

Integral and Peripheral Membrane Proteins

Proteins can be associated with the membrane in multiple ways. At the most fundamental level, proteins may

• exist solely on the surface of a membrane, living peripherally on one side or the other, or
• extend into the nonpolar tail region of the membrane in some way so that they are integrated into the membrane itself.

This may sound like a very simple thing to differentiate: Do they exist on the surface or extend into the membrane itself? However, it isn’t always as easy as it sounds. For example, how do we categorize a protein that sits on the surface but also has a covalently linked lipid tail that extends into the membrane? (Answer: We consider it to be integral.)

Since proteins in membranes are too small for us to see, the categorization of proteins has historically been based on experimental evidence. So if we include the experimental evidence in our definition, we come up with the following:

• Integral membrane proteins are proteins that cannot be removed from the membrane without destroying the membrane completely. Usually, this requires the use of strong detergents, which disrupt the structure of the membrane so that proteins can be removed. Only proteins that extend into the membrane in some way will require this level of disruption to extract.
• On the other hand, peripheral proteins are much easier to remove. Usually, a simple ionic salt wash is enough to dislodge these proteins from the membrane, as the intermolecular forces are not as strong and can be more easily disrupted.

Table 02-01: The difference between integral and peripheral membrane proteins

Criterion	Integral proteins	Peripheral proteins
Requirements for removal from the membrane	Strong detergents or organic solvents used to break up hydrophobic associations	Mild detergents, high salt concentrations, and/or metal chelating agents used to remove
Association with lipids after extraction	Usually remain partially associated with lipids after removal	Usually completely free of lipids once removed
Solubility after extraction	Usually insoluble or clumped together in neutral aqueous solutions (e.g., water)	Soluble and dispersed in neutral aqueous solutions

Figure 02-11: Examples of membrane proteins. (A) Integral transmembrane proteins. The protein on the left is a single alpha-helical region spanning the membrane. The protein on the right shows a beta barrel configuration. (B) An integral monolayer-associated protein. The alpha helix associates only with one leaflet of the membrane bilayer. (C) Peripheral membrane protein. The protein highlighted in pink is noncovalently attached to a membrane-anchored protein (gray). This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Figure 02-11 also helps illustrate the different subcategories of membrane proteins:

• Transmembrane proteins (Panel A): These integral membrane proteins cross the entire membrane and stick out on either side into the cytosol. These proteins are held in place primarily through the hydrophobic effect. More on that in the section below.
• Monolayer-associated proteins (Panel B): This is also a type of integral membrane protein. Although they are not considered to be very common, they still occur rarely. In this case, the protein is held in place by an alpha helix that is amphipathic (i.e., nonpolar on one side only). We will not be discussing this type further.
• Peripheral membrane proteins (Panel C): These proteins are most commonly (but not always) attached to the membrane through associations with integral membrane proteins. These linkages are most often via ionic or hydrogen bonding or some combination of several types of intermolecular forces. The association of peripheral proteins with one side of the membrane or the other further contributes to the asymmetry of the membrane.

Transmembrane Proteins Require Specific Secondary Structure to Be Able to Pass through the Membrane

One of the biggest challenges faced when proteins must pass through a membrane is how to deal with their backbones. Unlike the composition of R groups, the backbone of the polypeptide chain is always polar. The H, N, and O atoms that make up the peptide bond are electronegative and capable of forming hydrogen bonds. As such, they face a thermodynamic challenge when required to pass through the hydrophobic center of the lipid bilayer. So how is this addressed in the cell?

The answer lies in their secondary structure. As you may recall, secondary structures are defined as local, repeating structures that are formed via hydrogen bonding of backbone atoms to other backbone atoms. These repeating structures usually have a twofold effect:

• they allow the protein backbone to form hydrogen bonds with itself, which is thermodynamically stable, and
• they usually push the R groups outward, where they are available to interact directly with the environment. This leaves the protein backbone in the center, sequestered away from the nonpolar lipid environment.

The two secondary structures we mentioned earlier are the alpha helix and the beta sheet. Both of these structures are very commonly found in transmembrane proteins. However, the details of how they form are naturally going to be different.

Alpha Helices in Transmembrane Proteins

Many transmembrane proteins have one or more alpha helices in their transmembrane domains. These alpha helices are stretches of about 20+ nonpolar amino acids (depending on the width of the membrane). Remember that in the alpha-helical arrangement, the amino acid R groups extend outward, and the backbone is in the center of the helix. By forming an alpha helix, the nonpolar R groups will shield the polar polypeptide backbone from the nonpolar environment in the center of a lipid bilayer, thus creating thermodynamic stability. Figure 02-12 shows an alpha helix from the side, embedded in a membrane. You can see the backbone structure represented by the purple ribbon winding up through the membrane. Hydrogen bonds between the atoms of the backbone (shown as dashed lines between the ribbon) hold the shape of the helix. The R groups of each amino acid in the polypeptide chain extend outward from the polypeptide backbone to interact with the tails of the phospholipids.

Figure 02-12: Integral membrane protein with a single alpha helix crossing the membrane. The polypeptide backbone curls into a helix (purple ribbon), while the R groups (blue circles) of each amino acid in the chain extend into the nonpolar portion of the membrane. Dashed lines represent the hydrogen bonds. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Since transmembrane proteins must be able to exist in both the nonpolar center of the membrane and the aqueous parts of the cell, it stands to reason that these proteins are amphipathic like other components of the biological membranes. The R groups of amino acids within the nonpolar portion of the membrane are nonpolar, whereas in other regions that interact with the aqueous environments, the amino acids will primarily be polar.

It is important to note that a single alpha helix does not form a channel, so it cannot allow anything to pass through it. The molecules of the backbone completely fill the space inside the helix (Figure 02-13). Alpha helices can be used to form the pores and channels that control the entry/exit of many molecules in/out of the cell. Because a single helix cannot act as a channel, several membrane-spanning alpha helices must instead cluster in a roughly circular arrangement through the membrane.

Figure 02-13: Different top-down views of a single alpha helix. Ribbon models of helices (shown on the left, with the R groups sticking out the “ribbon” path of the backbone) can give a false illusion that there is space inside the helix large enough for molecules to pass through. The space-filling model confirms that the internal area of the helix is filled by the space taken up by the atoms of the peptide backbone. This image is a derivative of 5EH6 created with NGL viewer by Dr. Lauren Dalton and is shared under a CC BY-SA 4.0 license.

Aquaporin is an excellent example of a transmembrane protein that is made of several alpha helices, which are used to create a central channel (Figure 02-14). Aquaporin is nonselective and allows water and other small solutes to pass through the membrane. Note that the amino acids lining the interior of the channel will need to be polar and possibly charged in order to interact with the water and solutes that are expected to pass through. On the other hand, the exterior of aquaporin will have a large strip that will directly interact with the phospholipid tails of the membrane; thus, those regions will need to be nonpolar.

Figure 02-14: The protein structure of the water channel protein aquaporin, shown in multiple formats and angles. For simplicity, the R groups are not shown in the image, but they extend outward from the helical ribbon as we saw in Figure 02-13. The side view illustrates the orientation of the helical bundles to form a column through the membrane. The top views illustrate the circular arrangement that is made with enough room to form a small pore through the membrane. This image is a derivative of 1H6I created with NGL viewer by Dr. Lauren Dalton and is shared under a CC BY-SA 4.0 license.

Beta Barrels in Transmembrane Proteins

While a single alpha helix is stable enough to be used as the sole membrane-spanning structure for a transmembrane protein, a beta sheet is not as well designed for this purpose. The edges of the sheet will have exposed backbone molecules that will not easily interact with the nonpolar portion of the bilayer. However, a beta sheet can circularize itself by hydrogen bonding the ends of the sheet together (Figure 02-15). This means that the backbone’s bonding requirements are met within the nonpolar region of the membrane, and we once again have an ideal scenario for a thermodynamically stable structure that can pass through a membrane. We call these circularized beta sheet structures beta barrels.

Figure 02-15: Beta sheet structure within a beta barrel. The image on the left is a beta barrel embedded in a membrane. The pore formed in the center is lined with polar R groups (orange), while the membrane side is lined with nonpolar R groups (blue). The box on the right is a magnified view to show further detail of the beta barrel section highlighting the peptide backbone interactions and their relationship to the R groups in a beta barrel. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Like the alpha helix, beta sheets and beta barrels require a specific arrangement of their R groups in order to form. Unlike the alpha helix, the R groups stick out perpendicular to the face of the beta sheet in an alternating pattern (Figure 02-15). This means that if we were to unfold the beta barrel and examine the primary sequence, we’d likely see a series of amino acids with alternating properties (i.e., nonpolar, polar, nonpolar, etc.) because one amino acid with an R group facing the lipid environment would be next to an amino acid with an R group facing the aqueous pore environment.

We can see this in action by examining the beta barrel structure of a real bacterial protein called outer membrane protein G (Figure 02-16). Once again, we see that this particular secondary structure, the beta barrel, precisely suits the function of the protein as a transporter. This protein is used to take up large carbohydrates. Thus, the outside of the beta barrel must be able to interact with the lipid environment and help hold the protein in the membrane, whereas the inside of the barrel must create enough space to allow specific molecules to pass through the membrane. In addition, the amino acids of the center channel must be polar as well to interact with the carbohydrates that are transported. In summary, the properties of the amino acids must match the chemical environment where they reside and function.

Figure 02-16: Multiple views of bacterial outer membrane protein G. The side view illustrates the orientation of the beta sheets to form a barrel structure through the membrane. The top views illustrate the circular arrangement that is made with enough room to form a small pore through the membrane. Note that R groups are removed from this image for simplicity. This image is a derivative of 2JQY created with NGL viewer by Dr. Lauren Dalton and is shared under a CC BY-SA 4.0 license.

Function of Membrane Proteins

Membrane proteins carry out many different functions in the cell. It is important to remember that membranes in the cell have different sets of proteins in them, as they each must carry different functions. As such, the protein composition of the endoplasmic reticulum (ER) membrane is different from, say, the plasma membrane.

Figure 02-17: Examples of different functions of membrane proteins. Here we see linker proteins, anchors, transporters, receptors, and other enzymes that are embedded in the plasma membrane of a cell. See text for the details of the function of each of these. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

While there are many different functions for membrane proteins, for the most part they’ll fall into one of the following categories (Figure 02-17):

• Structural proteins, such as linkers and anchors—Anchors help attach the membrane to organelles, the extracellular matrix, or even other cells, whereas linkers help connect several proteins in the membrane and can help provide shape. We will see many examples of structural proteins as we discuss cellular function.
• Transporters—These proteins mediate the transport of different types of molecules across the membrane in either direction. We saw examples of these in our earlier discussion of alpha helices and beta barrels. We will look at some examples, such as proton pumps and the ATP synthase.
• Enzymes—Many membrane proteins have enzymatic activity for a whole variety of cellular functions. We will explore many examples, including synthesizing or modifying enzymes, flippases, scramblases (shown earlier, in Figure 02-07), or kinases.
• Receptors—Receptors are key for the cell to be able to sense and respond to its environment. Receptors extend across the membrane and bind to small molecules or other proteins on the outside of the cell and in response initiate a chain of events leading to transmission of a signal inside the cell. Many receptors are also enzymes. We will look at receptors in more detail when we discuss cell signaling.

The Plasma Membrane: An Example of a Real Biological Membrane

The plasma membrane is the membrane that surrounds the cell and is the first point of contact between the cell and its environment. Red blood cells were one of the first cells to have their plasma membranes studied and as such have one of the most well-characterized plasma membranes. In most cell types, the plasma membrane is supported and shaped by a network of proteins. In red blood cells, most of the protein network is inside the cell, just underneath the plasma membrane. However, in other cell types, this network can also be on the outside (via connections to the extracellular matrix or cell wall) or on both sides of the membrane.

Figure 02-18 shows a diagram of the plasma membrane of a red blood cell. In it, we see that the lipid bilayer of the plasma membrane is attached to an internal meshwork of spectrin protein filaments. These filaments provide a framework that supports the membrane and gives it its elasticity. You can also see that lipids and proteins on the extracellular side of the membrane are connected to carbohydrates (as indicated by the glyco- prefix). Since red blood cells move through the bloodstream, spectrin does not make any permanent connections with external structures.

Figure 02-18: The plasma membrane of a red blood cell. There are a variety of different proteins found in the plasma membrane of the red blood cell. Carbohydrate groups (purple squares) are found on the exterior side of the membrane and contribute to cell identity. On the inside, we see the actin network, connected to spectrin and ankyrin. Together, these proteins help give the red blood cell its characteristic concave shape. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Plasma Membrane Carbohydrate Groups Are Found on the Outside of the Cell

Something that is important to point out in the plasma membrane figures in this chapter (see Figures 02-06, 02-10, and 02-18 as examples) is that the exterior of the plasma membrane is usually covered in a coating of carbohydrates. These carbohydrates are most commonly in the form of glycolipids and glycoproteins that are integrated directly into the plasma membrane. The carbohydrate component of the plasma membrane has a very important function. In biological systems, cells have an identity (and can be recognized) on the basis of the configuration of carbohydrate molecules on the surface of the membrane.

A classic example of this is your blood type. The cell surface polysaccharides carried by your red blood cells are genetically determined so that your body knows which cells belong to you and which are foreign. The ability to identify foreign cells is absolutely vital for your body so that it can recognize pathogens and other invaders. This also influences our ability to carry out blood transfusions when needed. Your “blood type” makes direct reference to the polysaccharides carried on the surface of your cells. The ABO system is the most well known; however, there are other cell surface signals that the body uses to identify which blood belongs to you, such as the Rhesus (Rh) factor. If you receive a transfusion of blood that contains the incorrect polysaccharide markers on the cell surface, your body will produce antibodies to attack the blood and destroy it.

In some cell types (such as bacteria but also many eukaryotic cells), this carbohydrate coating on the plasma membrane is complex enough that it has its own name: the glycocalyx. Most examples of eukaryotic cells that have a glycocalyx are found in animals. This includes many epithelial cells (like the cells lining the gut and our blood vessels). Another really great example of a glycocalyx, which you can actually see with your naked eye, is the slimy coating on fish. The polysaccharide coating is found on virtually all fish, including the sockeye salmon shown in Figure 02-19. It plays multiple roles, including protection, cell-to-cell recognition, and even immune functions.

Figure 02-19: A male (bottom) and female (top) sockeye salmon (Oncorhynchus nerka). The glycocalyx is on the exterior of the body and adds to the shininess of the fish seen here. It feels slimy if you pick up a live or recently dead fish. This image originates from the National Digital Library of the United States Fish and Wildlife Service and is in the public domain.

Plasma Membrane–Adjacent Structures in Animal and Plant Cells

Cells in tissues are surrounded by a matrix of protein, polysaccharide, and fluid. The composition of the extracellular environment varies widely in different tissue types as well as in different organisms from different kingdoms. However, a common theme is that there are quite a lot of carbohydrates. The carbohydrates may or may not be associated with proteins, but collectively they form a three-dimensional network that connects cells together and provides a sort of hydration layer to trap water near the cells. Here we will highlight some of the key differences between this external environment of plant and animal cells.

In both plants and animals, the extracellular macromolecules are synthesized and secreted by the cells that live within them through a process called exocytosis, which we will cover in Chapter 4.

The Extracellular Matrix of Animal Cells

The extracellular matrix (ECM) of animal cells is made primarily of proteoglycans. These are very similar to glycoproteins, except there is quite a lot more polysaccharide attached to a relatively small protein. The proteins and polysaccharides of the extracellular matrix are all connected to each other in a large 3D network, making it difficult to tell where one molecule ends and another begins. Polysaccharide chains known as hyaluronic acid are a major component of the animal extracellular matrix. Hyaluronic acid is important due to its gel-like properties. It helps trap water, which makes the entire ECM look and feel a bit like Jell-O. Thus, polysaccharides like hyaluronic acid help the extracellular matrix remain hydrated and, as such, resist compression. For this reason, it is a large component of the cartilage in our joints.

Figure 02-20: Artist’s rendition of the mammalian extracellular matrix. Illustration by David S. Goodsell, RCSB Protein Data Bank, https://doi.org/10.2210/rcsb_pdb/goodsell-gallery-033. Shared under a CC BY 4.0 license.

The most abundant protein in the mammalian extracellular matrix (not to mention the most abundant protein in the entire human body) is a very large protein called collagen (Figure 02-20). This protein is made of three intertwined polypeptide chains, which makes collagen very strong. Collagen provides structural support and helps with things like wound healing.

The Plant Cell Wall

The plant cell wall is also made mostly of carbohydrates. Interestingly, it contains many fewer proteins and is more rigid than an animal extracellular matrix. There is a cell wall that surrounds every single cell in a plant. So while it can be thought of as a type of extracellular matrix for the plant, its role is more complex than that of the animal extracellular matrix. It provides the structural support for the whole plant, similar to the role of the skeleton in animals.

Like the animal extracellular matrix, the plant cell wall is synthesized and secreted by the cell. Most of the polysaccharide components are synthesized by the Golgi apparatus; however, there is one notable exception. Cellulose is a very strong fiber of crystallized glucose chains that is synthesized in a unique structure called a rosette (Figure 02-21). This rosette is embedded in the plasma membrane, and it moves through the membrane as cellulose is synthesized. Microtubules have a role to play in the synthesis of cellulose and, as such, have a very different organization than we see in animal cells.

While there are a few proteoglycans and glycoproteins in the plant cell wall, the complex, branched polysaccharides are really the key players in both its structure and its function. In addition to cellulose, there are other long polysaccharide chains, such as hemicellulose (used to connect the cellulose together) and pectin. Pectin acts in a similar way to hyaluronic acid in that it helps trap water and create a gel matrix. Incidentally, this is also why we use pectin to make jam.

Figure 02-21: Artist’s rendition of the plant cell wall and its underlying cell. Illustration by David S. Goodsell, RCSB Protein Data Bank, https://doi.org/10.2210/rcsb_pdb/goodsell-gallery-029. Shared under a CC BY 4.0 license.