An Introduction to Biotechnology: The Science, Technology and Medical Applications

engineering.

Chapter 1

Membranes

Abstract

All biotechnologies involve cells at some level. Common biotechnologies entail communication or manipulation of cells, often by the delivery of drugs, signaling molecules, or other bioactive agents. In order for such agents to operate, interaction with the cell membrane will have to occur. The cell membrane separates the inside of the cell from the extracellular environment. Signals can cross the membrane either by traversing the membrane or by interacting with the extracellular face of the membrane and having the event indicated somehow on the cytoplasmic face of the cell. Before we deliver a drug or a signal to a cell, we should first understand what the cell membrane is. In this chapter, we will discuss the composition and function of the plasma membrane.

Keywords

Lipid

Fatty acid

Phospholipid

Amphipathic

Phosphatidylethanolamine

Phosphatidylserine

Phosphatidylcholine

Cholesterol

Protein

The biotechnologist will inevitably have to deal with cells at some point. Whether an extracellular matrix is being isolated to serve as a tissue engineering scaffold, a gene is being delivered to treat a genetic disease, or a biofuel is being produced as a type of renewable energy, cells will be involved during the research and development process (if not the final application of the technology itself). A basic understanding of the cell is therefore critical to the development of biotechnologies.

We will begin our survey of the cell with (one of) the exterior layer(s) of the cell—the plasma membrane. At the cellular level, this is the first point of contact for technologies such as immunotherapy, gene delivery, and patterned cell attachment. When designing a particle that is to be taken up by a cell, a thorough knowledge of the plasma membrane is advisable.

1.1 Membrane Lipids

Both eukaryotic and prokaryotic cells are surrounded by a plasma membrane (Figure 1.1). The plasma membrane is not the outermost layer of a prokaryotic cell, but this point will be addressed in detail later in this chapter. The plasma membrane is made up of lipids, carbohydrates, and proteins, and various combinations thereof, but the primary constituent is the phospholipid. There are many different types of phospholipids, but the ones used in the plasma membrane all follow some basic principles.

Figure 1.1 The eukaryotic plasma membrane.

Membrane phospholipids contain a hydrophilic head and two hydrophobic tails, and as such are said to be amphipathic. Phospholipids can be considered as three constituents linked to a glycerol backbone via reactions with each of the three hydroxyls. The structure of glycerol is given in Figure 1.2.

Figure 1.2 The structure of glycerol, the backbone of the phospholipid structure.

Of course, there are exceptions, but the following rules of thumb help to describe the structure of a phospholipid built off of a glycerol backbone:

1. The first hydroxyl will have been used to react with the C-terminus of a fatty acid to form an ester linkage. The fatty acid is typically 14-24 carbons long, with an even number of carbons. The fatty acid will be saturated.

2. The second hydroxyl will also be attached to a fatty acid with an even number (14-24) of carbons in an ester linkage. The fatty acid differs from the first one in that it is typically unsaturated. The unsaturated fatty acid will be kinked, which results in less dense packing in the membrane and therefore a lower freezing temperature.

3. The third hydroxyl will be linked to a phosphate to form an ester bond. The arm of the molecule that contains the phosphate group will constitute the hydrophilic portion of the phospholipid. If this arm has only a phosphate group, the lipid is phosphatidic acid (Figure 1.3). However, additional moieties can be attached to the phosphoryl group to give rise to the range of phospholipids found in the plasma membrane.

Figure 1.3 Phosphatidic acid.

Three of the most common phospholipids of the plasma membrane are phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. Figure 1.4 gives general structures of these phospholipids, but it can also be seen that there is not one single formula for a given phospholipid. The compositions of the two fatty acids attached to the glycerol backbone are not fixed, which means that there can be several different phosphatidylcholines, for example.

Figure 1.4 Structures, charges, and general locations of three of the most common phospholipids in the plasma membrane.

There are two additional types of lipids that are common members of the plasma membrane that bear mention. The first is sphingomyelin, a phospholipid that has a choline head group but differs from phosphatidylcholine in that instead of a glycerol backbone holding two fatty acid tails, the hydrophobic portion of the molecule is ceramide (Figure 1.5). As the name implies, sphingomyelin is found in the myelin sheath that surrounds the axons of certain nerve cells, but it also makes up a significant percentage of the plasma membranes of cells such as liver and red blood cells.

Figure 1.5 Sphingomyelin.

Glycolipids also bear mention because of their prevalence in certain plasma membranes. Regarding the plasma membranes of animal cells, they are found only on the exterior surface, which implies that they may have some function in cell-cell interactions, cellular identification, or cell signaling. Although the glycolipid composition of the plasma membrane differs greatly between cells of different species or even between tissues in the same animal, it is believed that all cells contain at least some glycolipids in the exterior face of their plasma membranes. This is a feature that has yet to be fully capitalized upon in fields such as stem cell engineering or tissue engineering, although the medical science and immunology disciplines have recognized the glycolipid signatures of various microbes and have developed treatments accordingly.

The plasma membrane is asymmetrical due to the preferential locations of the above phospholipids to one side of the membrane or the other. As a general rule, the phospholipids with a terminal amino group tend to be on the inner (cytoplasmic) face of the plasma membrane (PE and PS), while those with a choline in their head group (PC and sphingomyelin) are typically found on the external face. Glycolipids are effectively always on the external face (see Figure 1.6a). These guidelines do not dictate the absolute positions of membrane constituents, however—the plasma membrane is a fluid structure. Molecules on the outer face can generally move about like people on the second floor of a crowded mall, and molecules on the inner face can move around like people on the first floor. Some lipids will stay together (in lipid rafts) much like how a family might stay together in the mall, while other lipids can change the side of the membrane they are on like somebody taking the escalator. Such translocation can be seen in specific circumstances, such as when a membrane is being actively formed by the addition of new lipids, as takes place in the endoplasmic reticulum. Enzymes known as phospholipid translocators cause the phenomenon, which is known as flip-flop. Another example of phospholipid flipping occurs during the process of programmed cell death known as apoptosis. During the early stages of this complex process, an enzyme known as scramblase randomizes phospholipid locations relative to which side of the membrane they usually reside (Figure 1.6b). One result is that PS will be flipped to the exterior face of the plasma membrane. The sudden appearance of these negatively charged phospholipids on the exterior portion of the cell is thought to serve as a signal to macrophages to engulf and degrade the apoptotic cells.

Figure 1.6 (a) A representative distribution of membrane phospholipids. (b) During apoptosis, scramblase randomizes phospholipid distributions. The redistribution of PS can be detected by other cells, such as macrophages.

1.2 Cholesterol

Cholesterol is another constituent of the plasma membrane. The structure of cholesterol is given in Figure 1.7. Although it is not a phospholipid, cholesterol has a similar structure in that it has a hydrophilic portion (the OH group) and a hydrophobic region (the rest of the molecule). Cholesterol fits between adjacent phospholipids in the plasma membrane, with the hydroxyl aligning itself with polar head groups and the rest of the molecule fitting in among the fatty acid tails (Figure 1.8). Cholesterol can be found in high abundance in animal cells, sometimes at concentrations of one cholesterol molecule for every phospholipid molecule. While many people are aware that high cholesterol content in the blood is considered a bad thing, cholesterol is nevertheless a necessary component of the cell membrane of every cell because of the several functions it serves. First, the four steroid rings are relatively rigid, which serves to decrease the fluidity of the membrane and increase its stability. With the hydroxyl group being positioned as it is, the rings will be firmly placed in the area of the glycerol backbones and first several carbons of the fatty acid tails of adjacent phospholipids. This effectively immobilizes those carbons of the adjacent phospholipids and renders the membrane less fluid. Second, while making the membrane less fluid, cholesterol also serves to make the membrane more flexible. While the lower-numbered carbons of phospholipid fatty acid tails are held in relative rigidity, the higher-numbered carbons (the furthest from the glycerol backbone) are free to gyrate. With cholesterol serving as a spacer, there is less chance for the carbons at the end of the tails of one phospholipid to associate with the tails of adjacent phospholipids. As a result, fatty acid tails are prevented from coming together to form ordered, crystalline structures, as will happen during phase transitions at lower temperatures (freezing).

Figure 1.7 The structure of cholesterol.

Figure 1.8 Cholesterol fits between adjacent phospholipids.

1.3 Membrane Proteins

A discussion of the plasma membrane would not be complete without a presentation of membrane proteins. However, membrane proteins cannot be adequately described without an introduction to proteins in general. Entire careers have been devoted to proteins, and many wonderful books exist devoted to the subjects of protein structure and function. While an exhaustive introduction to proteins is not appropriate for an introduction to biotechnology, a rudimentary knowledge of proteins is indeed required when learning about the structure and function of the cell. We will begin our survey in the next chapter with the building block of the protein: the amino acid.

Questions

1. What does cholesterol do for the cell membrane?

2. A phospholipid head has a nitrogen group attached to four carbons. Can it be found on the inside or outside layer of the plasma membrane, or both? Explain your answer.

3. Describe the usual structure of a phospholipid built off of a glycerol backbone.

4. What is the most common molecule found in the plasma membrane?

5. Why do phospholipids in solution form micelles?

6. Which of the above phospholipids might be a common constituent in the cell membrane? For the ones that are not expected to be common, give a reason why not.

7. Why is the charge on phosphatidylserine useful?

Chapter 2

Proteins

Abstract

In our journey into the cell, we will encounter membrane proteins. However, proteins are found throughout the cell, as well as outside the cell. Being such important constituents of the cell and biological processes, proteins are addressed as a stand-alone chapter. In fact, many of the signals delivered to cells are proteins, and many of the cellular products that a biotechnologist might want to harvest are proteins.

In this chapter, we will learn about amino acids, the building blocks of proteins, and then discuss molecular structures of proteins starting at a rudimentary level and progressing in complexity to fully folded, multi-subunit structures. The chapter ends with a return to membrane structure and how proteins play a role in membranes.

Keywords

Amino acid

pKa value

Isoelectric point

Primary, secondary, super-­secondary, tertiary, and quaternary protein structures

Hydrophobic effect

Conformation

Enzyme

Active site

Surfactant

Detergent

Soap

Denature

Peptide bond

α-Helix

β-Pleated sheet

β-Turn

2.1 Amino Acids

Amino acids are so named because they contain both an amino group and a carboxylic acid. The structure of an amino acid at neutral pH (pH = 7.0) is given in Figure 2.1. Disregarding the side group R for a moment, the figure shows that the molecule contains both a positive charge and a negative charge, making it a zwitterion. A zwitterion is a molecule that has both positive and negative regions of charge. In the common amino acids, the carbon that connects the amino and carboxylate groups will be chiral if the R group is anything except a hydrogen atom. Chirality is used to describe the property of certain molecules to rotate polarized light, with l-forms rotating polarized light to the left (counterclockwise) and d-forms rotating it to the right. Examples of l- and d-forms of an amino acid are given in Figure 2.2. Note that the two forms are mirror images of one another. While chirality may be thought of as interesting only to the organic chemist, it is important to the biotechnologist as well. In the case of amino acids, it is the l-form that is used in constructing proteins such as enzymes, receptors, and signaling peptides. d-form amino acids are sometimes used, with varying success, to produce biologically inert molecules.

Figure 2.1 The structure of an amino acid at neutral pH.

Figure 2.2 l -(top) and d -forms of the same amino acid. Heavier fonts indicate atoms coming toward the reader out of the plane of the page, while lighter fonts indicate atoms oriented away from the reader below the plane of the page.

While all amino acids have the basic structure shown in Figure 2.1, the identity of the R group defines each specific molecule. There are 20 amino acids commonly found in nature to form proteins, each with a defined R group. These amino acids are shown in Figure 2.3.

Figure 2.3 Structures, names, three-letter abbreviations, and one-letter codes of the common 20 amino acids used to form proteins. These formulas reflect the prevailing ionization states at pH = 7.0. The shaded portions of the structure are the entities common to each amino acid and would form the backbone in a polypeptide; the R group of each amino acid is unshaded. Because the p K a of the histidine R group is relatively close to 7.0, both ionization states are shown (with a hydrogen and positive charge in parentheses). The unionized form of the histidine R group will predominate at pH = 7.0. Circles around single-letter codes indicate nonpolar side groups. The circle around G is dashed because, although the small hydrogen side group is most easily classified as nonpolar, it contributes very little to hydrophobic interactions. Also note that C is considered polar here, although the side group is commonly bound to another C via a disulfide linkage, which is strongly nonpolar.

After examining Figure 2.3 and the accompanying figure caption, a couple of points may need further clarification. First, notice that aspartate and asparagine are grouped together, as are glutamate and glutamine. The difference between each pair of side groups is that the negatively charged oxygen has been replaced by an amine group (NH2), which is hydrophilic. These amino acids are paired together because (1) aspartate and asparagine are very similar chemically, as are glutamate and glutamine, and (2) they are all hydrophilic. Appearing on the second page of the figure are the positively charged amino acids, which are hydrophilic, followed by the aromatics, which tend to be hydrophobic. A case can be made that tyrosine can be considered hydrophilic because of the hydroxyl group on the aromatic ring. This is the reason that the one-letter code Y is not circled for tyrosine. Note that the other hydroxyl-containing amino acids are also hydrophilic.

Cysteine is a special amino acid in that its side group contains a sulfhydryl group. In a protein, oftentimes, cysteines will occur in pairs. The paired cysteines do not have to be next to each other (or even close to each other) in terms of the order of amino acids in a polypeptide. However, when cysteines pair in three dimensions as a part of protein folding, they will be in close proximity and form a disulfide bond. The pair of bonded cysteines will be known as a cystine (note the spellings). The disulfide bonds can be broken via reduction, which means that acidic environments or proton donors can convert cystines back into a pair of cysteines. As we will see, this is a property one must contend with in the laboratory, especially in terms of identifying the primary sequence of a protein.

Even though biotechnology students might at first be overwhelmed with the prospect of learning the structures of all 20 common amino acids, they should nevertheless strive to acquaint themselves with the structures and functions of these molecules. At the very least, they should have a working knowledge of the different classes of amino acids as they delve into different biotechnical applications. For instance, if one were interested in looking at specific amino acids for their propensity to bind with DNA or perhaps serve as a delivery vehicle for siRNA, negatively charged side groups (D and E) should be avoided because they would be repelled by the negative charges in the DNA or RNA molecules. Similarly, using a polypeptide made up entirely of nonpolar and uncharged amino acids (G, A, V, L, I, P, F, W, and M) for the purpose of a signaling molecule to be delivered via the blood would not be advisable because the molecules would be hydrophobic and therefore not soluble in the aqueous environment of the blood. Any sort of protein engineering should be accompanied by the knowledge of which amino acids are small, large, inflexible, charged, polar, hydrophobic, or cross-linkable.

2.1.1 pKa

COOH. The naming convention has the acid form as the form with its complete set of protons. When a proton is lost and the species carries a negative charge, we refer to this as the ate form, hence aspartate, as opposed to aspartic acid.

Not all acids ionize at the same pH. A way to characterize different acids with respect to the pH at which they will tend to lose a proton is known as the pKa value. Without deriving it, the pKa value is related to the dissociation constant. The definition of pKa is the pH at which an ionizable species is 50% ionized. Considering the side group for aspartate/aspartic acid, the pKa is 3.65. This implies that if one has a beaker of aspartate/aspartic acid in an aqueous, buffered solution held at pH 3.65, half of these amino acid molecules will be aspartate and half will be aspartic acid. What this tells us is that for aspartate/aspartic acid at physiological pH (7.2 inside the cell), the pH is a good distance from the pKa value on the number line, so virtually all of these molecules will be in the form of aspartate. As a general rule, a good distance in these cases generally refers to being at least one pH unit away from the pKa. The pKa value of aspartic acid being 3.65 does not imply that at pH = 3.66, all of these molecules will be in the form of aspartate; one would expect that slightly more than 50% would be. As the pH is increased further from the pKa value, a greater percentage of the side groups would be in the ate form over the acid form. In the body, changes in pH by 0.2 units can have profound effects on protein folding.

When deciding on the ionization state of a species, a handy rule to keep in mind is that at pH values below the pKa for that species, there will be a relative abundance of protons relative to the species, and it will carry as many protons as possible. At pH values above the pCOOH) form. At pH = 1, the amino acid Asp would be in the form of aspartic acid. The same is true for Glu, for which the side group has a pKa of 4.25, so the amino acid would be in the form of glutamic