Cells: The Fundamental Units of Life - PDF

PANEL 1–2 CELL ARCHITECTURE 25 ANIMAL CELL centrosome with pair of cent...

PANEL 1–2 CELL ARCHITECTURE 25 ANIMAL CELL centrosome with pair of centrioles microtubule chromatin (DNA) extracellular matrix nuclear pore vesicles lysosome mitochondrion 5 µm actin filaments nucleolus endoplasmic nucleus reticulum peroxisome plasma membrane ribosomes in Golgi intermediate cytosol apparatus filaments Golgi apparatus nucleolus mitochondrion Three cell types are drawn here in a more realistic manner than in the schematic drawing in Figure 1–24. The animal cell drawing is based on a fibroblast, a cell chromatin (DNA) flagellum that inhabits connective tissue and deposits extracellular nuclear pore matrix. A micrograph of a living fibroblast is shown in cell wall Figure 1–7A. The plant cell drawing is typical of a young leaf cell. The bacterium shown microtubule is rod-shaped and has a single flagellum for motility. A comparison of the scale bars vacuole ribosomes in (fluid-filled) reveals the bacterium’s cytosol relatively small size. outer membrane peroxisome DNA chloroplast plasma membrane ribosomes cell wall in cytosol BACTERIAL CELL PLANT CELL actin filaments lysosome 1 µm 5 µm 26 CHAPTER 1 Cells: The Fundamental Units of Life Figure 1–29 Where did eukaryotes nonphotosynthetic photosynthetic plants animals fungi archaea come from? The eukaryotic, bacterial, bacteria bacteria and archaean lineages diverged from one another more than 3 billion years ago— very early in the evolution of life on Earth. Some time later, eukaryotes are thought chloroplasts to have acquired mitochondria; later still, a subset of eukaryotes acquired chloroplasts. Mitochondria are essentially the same in single-celled eukaryote plants, animals, and fungi, and therefore TIME were presumably acquired before these mitochondria lines diverged about 1.5 billion years ago. bacteria archaea ancestral prokaryote hurly-burly, so as to allow more delicate and complex control of the way the cell reads out its genetic information. Such a primitive eukaryotic cell, with a nucleus and cytoskeleton, was most likely the sort of cell that engulfed the free-living, oxygen-consum- ing bacteria that were the likely ancestors of the mitochondria (see Figure 1–19). This partnership is ECB5 thought to have been established 1.5 billion e1.28/1.29 years ago, when the Earth’s atmosphere first became rich in oxygen. A subset of these cells later acquired chloroplasts by engulfing photosyn- thetic bacteria (see Figure 1–21). The likely history of these endosymbiotic events is illustrated in Figure 1–29. That single-celled eukaryotes can prey upon and swallow other cells is borne out by the behavior of many present-day protozoans: a class of free-living, motile, unicellular organisms. Didinium, for example, is a large, carnivorous protozoan with a diameter of about 150 μm—roughly 10 times that of the average human cell. It has a globular body encircled by two fringes of cilia, and its front end is flattened except for a single protrusion rather like a snout (Figure 1–30A). Didinium swims at high speed by means of its beating cilia. When it encounters a suitable prey, usually another type of protozoan, it releases numerous small, para- lyzing darts from its snout region. Didinium then attaches to and devours Figure 1–30 One protozoan eats another. (A) The scanning electron micrograph shows Didinium on its own, with its circumferential rings of beating cilia and its “snout” at the (A) top. (B) Didinium is seen ingesting another 100 µm ciliated protozoan, a Paramecium, artificially colored yellow. (Courtesy of D. Barlow.) (B)  Model Organisms 27 (C) (D) (A) (B) (E) (F) (G) the other cell, inverting like a hollow ball to engulf its victim, which can Figure 1–31 An assortment of protozoans be almost as large as itself (Figure 1–30B). illustrates the enormous variety within this class of single-celled eukaryotes. Not all protozoans are predators. They can be photosynthetic or carnivo- These drawings are done to different scales, rous, motile or sedentary. Their anatomy is often elaborate and includes but in each case the scale bar represents such structures as sensory bristles, photoreceptors, beating cilia, stalklike 10 μm. The organisms in (A), (C), and (G) are ciliates; (B) is a heliozoan; (D) is an amoeba; appendages, mouthparts, stinging darts, and musclelike contractile bun- (E) is a dinoflagellate; and (F) is a euglenoid. dles. Although they are single cells, protozoans can be as intricate and To see the latter in action, watch Movie 1.6. versatile as many multicellular organisms (Figure 1–31). Much remains Because these organisms can only be seen to be learned about fundamental cell biology from studies of these fasci- with the aid of a microscope, they are also nating life-forms. referred to as microorganisms. (From M.A. ECB5 e1.30/1.31 Sleigh, The Biology of Protozoa. London: Edward Arnold, 1973. With permission from Edward Arnold.) MODEL ORGANISMS All cells are thought to be descended from a common ancestor, whose fundamental properties have been conserved through evolution. Thus, knowledge gained from the study of one organism contributes to our understanding of others, including ourselves. But certain organisms are easier than others to study in the laboratory. Some reproduce rapidly and are convenient for genetic manipulations; others are multicellular but transparent, so the development of all their internal tissues and organs can be viewed directly in the live animal. For reasons such as these, biol- ogists have become dedicated to studying a few chosen species, pooling their knowledge to gain a deeper understanding than could be achieved if their efforts were spread over many different species. Although the roster of these representative organisms is continually expanding, a few stand out in terms of the breadth and depth of information that has been accu- mulated about them over the years—knowledge that contributes to our understanding of how all cells work. In this section, we examine some of these model organisms and review the benefits that each offers to the study of cell biology and, in many cases, to the promotion of human health. Molecular Biologists Have Focused on E. coli In molecular terms, we understand the workings of the bacterium Escherichia coli—E. coli for short—more thoroughly than those of any other living organism (see Figure 1–11). This small, rod-shaped cell nor- mally lives in the gut of humans and other vertebrates, but it also grows happily and reproduces rapidly in a simple nutrient broth in a culture bottle. 28 CHAPTER 1 Cells: The Fundamental Units of Life Most of our knowledge of the fundamental mechanisms of life—including how cells replicate their DNA and how they decode these genetic instruc- tions to make proteins—has come from studies of E. coli. Subsequent research has confirmed that these basic processes occur in essentially the same way in our own cells as they do in E. coli. Brewer’s Yeast Is a Simple Eukaryote We tend to be preoccupied with eukaryotes because we are eukaryotes ourselves. But humans are complicated and reproduce slowly. So to get a handle on the fundamental biology of eukaryotes, we study a simpler representative—one that is easier and cheaper to keep and reproduces more rapidly. A popular choice has been the budding yeast Saccharomyces 10 µm cerevisiae (Figure 1–32)—the same microorganism that is used for brew- ing beer and baking bread. Figure 1–32 The yeast Saccharomyces cerevisiae is a model eukaryote. In this S. cerevisiae is a small, single-celled fungus that is at least as closely scanning electron micrograph, a number related to animals as it is to plants. Like other fungi, it has a rigid cell wall, of the cells are captured in the process of dividing, which they do by budding. is relatively immobile, and possesses mitochondria but not chloroplasts. Another micrograph of the same species When nutrients are plentiful, S. cerevisiae reproduces almost as rapidly as is shown in Figure 1–14. (Courtesy of Ira a bacterium. Yet it carries out all the basic tasks that every eukaryotic cell Herskowitz and Eric Schabtach.) must perform. Genetic and biochemical studies in yeast have been crucial ECB5 e1.31/1.32 to understanding many basic mechanisms in eukaryotic cells, including the cell-division cycle—the chain of events by which the nucleus and all the other components of a cell are duplicated and parceled out to create two daughter cells. The machinery that governs cell division has been so well conserved over the course of evolution that many of its components can function interchangeably in yeast and human cells (How We Know, pp. 30–31). Darwin himself would no doubt have been stunned by this dramatic example of evolutionary conservation. Arabidopsis Has Been Chosen as a Model Plant The large, multicellular organisms that we see around us—both plants and animals—seem fantastically varied, but they are much closer to one another, in their evolutionary origins and their basic cell biology, than they are to the great host of microscopic single-celled organisms. Whereas bacteria, archaea, and eukaryotes separated from each other more than 3 billion years ago, plants, animals, and fungi diverged only about 1.5 billion years ago, and the different species of flowering plants less than 200 million years ago (see Figure 1–29). The close evolutionary relationship among all flowering plants means that we can gain insight into their cell and molecular biology by focusing on just a few convenient species for detailed analysis. Out of the several hundred thousand species of flowering plants on Earth today, molecular biologists have focused their efforts on a small weed, the common wall cress Arabidopsis thaliana (Figure 1–33), which can be grown indoors in large numbers: one plant can produce thousands of offspring within 8–10 weeks. Because genes found in Arabidopsis have counterparts in agricultural species, studying this simple weed provides insights into the development and physiology of the crop plants upon which our lives depend, as well as into the evolution of all the other plant species that dominate nearly every ecosystem on the planet. Figure 1–33 Arabidopsis thaliana, the common wall cress, is a model plant. This small weed has become the favorite organism of plant molecular and developmental biologists. (Courtesy of Toni 1 cm Hayden and the John Innes Centre.)  Model Organisms 29 Figure 1–34 Drosophila melanogaster is a favorite among developmental biologists and geneticists. Molecular genetic studies on this small fly have provided a key to the understanding of how all animals develop. (Edward B. Lewis. Courtesy of the Archives, California Institute of Technology.) 1 mm Model Animals Include Flies, Worms, Fish, and Mice Multicellular animals account for the majority of all named species of living organisms, and the majority of animal species are insects. It is fit- QUESTION 1–7 ting, therefore, that an insect, the small fruit fly Drosophila melanogaster (Figure 1–34), should occupy a central place in biological research. The Your next-door neighbor has foundations of classical genetics (which we discuss in Chapter 19) were donated $100 in support of cancer ECB5 e1.33/1.34 research and is horrified to learn built to a large extent on studies of this insect. More than 80 years ago, that her money is being spent on genetic analysis of the fruit fly provided definitive proof that genes—the studying brewer’s yeast. How could units of heredity—are carried on chromosomes. In more recent times, you put her mind at ease? Drosophila, more than any other organism, has shown us how the genetic instructions encoded in DNA molecules direct the development of a ferti- lized egg cell (or zygote) into an adult multicellular organism containing vast numbers of different cell types organized in a precise and predict- able way. Drosophila mutants with body parts strangely misplaced or oddly patterned have provided the key to identifying and characterizing the genes that are needed to make a properly structured adult body, with gut, wings, legs, eyes, and all the other bits and pieces—all in their cor- rect places. These genes—which are copied and passed on to every cell in the body—define how each cell will behave in its social interactions with its sisters and cousins, thus controlling the structures that the cells can create, a regulatory feat we return to in Chapter 8. More importantly, the genes responsible for the development of Drosophila have turned out to be amazingly similar to those of humans—far more similar than one would suspect from the outward appearances of the two species. Thus the fly serves as a valuable model for studying human development as well as the genetic basis of many human diseases. Another widely studied animal is the nematode worm Caenorhabditis elegans (Figure 1–35), a harmless relative of the eelworms that attack the Figure 1–35 Caenorhabditis elegans is a small nematode worm that normally lives in the soil. Most individuals are hermaphrodites, producing both sperm and eggs (the latter of which can be seen just beneath the skin along the underside of the animal). C. elegans was the first multicellular organism to have its complete genome 0.2 mm sequenced. (Courtesy of Maria Gallegos.) 32 CHAPTER 1 Cells: The Fundamental Units of Life roots of crops. Smaller and simpler than Drosophila, this creature devel- ops with clockwork precision from a fertilized egg cell into an adult that has exactly 959 body cells (plus a variable number of egg and sperm cells)—an unusual degree of regularity for an animal. We now have a minutely detailed description of the sequence of events by which this occurs—as the cells divide, move, and become specialized according to strict and predictable rules. And a wealth of mutants are available for testing how the worm’s genes direct this developmental ballet. Some 70% of human genes have some counterpart in the worm, and C. elegans, like Drosophila, has proved to be a valuable model for many of the devel- opmental processes that occur in our own bodies. Studies of nematode development, for example, have led to a detailed molecular understand- ing of apoptosis, a form of programmed cell death by which animals (A) 1 cm dispose of surplus cells, a topic discussed in Chapter 18. This process is also of great importance in the development of cancer, as we discuss in Chapter 20. Another animal that is providing molecular insights into developmen- tal processes, particularly in vertebrates, is the zebrafish (Figure 1–38A). (B) 1 mm Because this creature is transparent for the first two weeks of its life, it provides an ideal system in which to observe how cells behave during Figure 1–38 Zebrafish are popular models development in a living animal (Figure 1–38B). for studies of vertebrate development. (A) These small, hardy, tropical fish—a staple Mammals are among the most complex of animals, and the mouse has in many home aquaria—are easy and cheap long been used as the model organism in which to study mammalian to breed and maintain. (B) They are also genetics, development, immunology, and cell biology. Thanks to mod- ideal for developmental studies, as their ern molecular biological techniques, it is possible to breed mice with transparent embryos develop outside the mother, making it easy to observe cells deliberately engineered mutations in any specific gene, or with artificially moving and changing their characters in constructed genes introduced into them (as we discuss in Chapter 10). the living organism as it develops. In this In this way, one can test what a given gene is required for and how it image of a two-day-old embryo, taken with functions. Almost every human gene has a counterpart in the mouse, a confocal microscope, a green fluorescent with a similar DNA sequence and function. Thus, this animal has proven protein marks the developing lymphatic vessels and a red fluorescent protein marks an excellent model for studying genes that are important in both human developing blood vessels; regions where health and disease. the two fluorescent markers coincide appear yellow. (A, courtesy of Steve Baskauf; Biologists Also Directly Study Humans and Their Cells B, from H.M. Jung et al., Development 144:2070–2081, 2017.) Humans are not mice—or fish or flies or worms or yeast—and so many scientists also study human beings themselves. Like bacteria or yeast, our individual cells can be harvested and grown in culture, where inves- tigators can study their biology and more closely examine the genes that govern their functions. Given the appropriate surroundings, many human cell types—indeed, many cell types of animals or plants—will survive, proliferate, and even express specialized properties in a culture dish. Experiments using such cultured cells are sometimes said to be carried ECB5 e1.37/1.38 out in vitro (literally, “in glass”) to contrast them with experiments on intact organisms, which are said to be carried out in vivo (literally, “in the living”). Although not true for all cell types, many cells—including those harvested from humans—continue to display the differentiated properties appropri- ate to their origin when they are grown in culture: fibroblasts, a major cell type in connective tissue, continue to secrete proteins that form the extra- cellular matrix; embryonic heart muscle cells contract spontaneously in the culture dish; nerve cells extend axons and make functional connec- tions with other nerve cells; and epithelial cells join together to form continuous sheets, as they do inside the body (Figure 1–39 and Movie 1.7). Because cultured cells are maintained in a controlled environment, they are accessible to study in ways that are often not possible in vivo. For example, cultured cells can be exposed to hormones or growth factors,  Model Organisms 33 (A) (B) (C) 50 µm 50 µm 50 µm Figure 1–39 Cells in culture often display properties that reflect their origin. These phase-contrast micrographs show a variety of cell types in culture. (A) Fibroblasts from human skin. (B) Human neurons make connections with one another in culture. (C) Epithelial cells from human cervix form a cell sheet in culture. (Micrographs courtesy of ScienCell Research Laboratories, Inc.) and the effects that these signal molecules have on the shape or behavior of the cells can be easily explored. Remarkably, certain human embryo cells can be coaxed into differentiating into multiple cell types, which can self-assemble into organlike structures that closely resemble a nor- mal organ such as an eye or brain. Such organoids can be used to study ECB5 n1.101/1.39 developmental processes—and how they are derailed in certain human genetic diseases (discussed in Chapter 20). In addition to studying our cells in culture, humans are also examined directly in clinics. Much of the research on human biology has been driven by medical interests, and the medical database on the human species is enormous. Although naturally occurring, disease-causing mutations in any given human gene are rare, the consequences are well documented. This is because humans are unique among animals in that they report and record their own genetic defects: in no other species are billions of individuals so intensively examined, described, and investigated. Nevertheless, the extent of our ignorance is still daunting. The mamma- lian body is enormously complex, being formed from thousands of billions of cells, and one might despair of ever understanding how the DNA in a fertilized mouse egg cell directs the generation of a mouse rather than a fish, or how the DNA in a human egg cell directs the development of a human rather than a mouse. Yet the revelations of molecular biology have made the task seem eminently approachable. As much as anything, this new optimism has come from the realization that the genes of one type of animal have close counterparts in most other types of animals, apparently serving similar functions (Figure 1–40). We all have a com- mon evolutionary origin, and under the surface it seems that we share the same molecular mechanisms. Flies, worms, fish, mice, and humans thus provide a key to understanding how animals in general are made and how their cells work. Comparing Genome Sequences Reveals Life’s Common Heritage At a molecular level, evolutionary change has been remarkably slow. We can see in present-day organisms many features that have been preserved through more than 3 billion years of life on Earth—about one- fifth of the age of the universe. This evolutionary conservatism provides 34 CHAPTER 1 Cells: The Fundamental Units of Life Figure 1–40 Different species share similar genes. The human baby and the mouse shown here have remarkably similar white patches on their foreheads because they both have defects in the same gene (called Kit), which is required for the normal development, migration, and maintenance of some skin pigment cells. (Courtesy of R.A. Fleischman, Proc. Natl. Acad. Sci. U.S.A. 88:10885–10889, 1991.) the foundation on which the study of molecular biology is built. To set the scene for the chapters that follow, therefore, we end this chapter by ECB5 e1.39/1.40 considering a little more closely the family relationships and basic simi- larities among all living things. This topic has been dramatically clarified by technological advances that have allowed us to determine the com- plete genome sequences of thousands of organisms, including our own species (as discussed in more detail in Chapter 9). The first thing we note when we look at an organism’s genome is its over- all size and how many genes it packs into that length of DNA. Prokaryotes carry very little superfluous genetic baggage and, nucleotide-for-nucleo- tide, they squeeze a lot of information into their relatively small genomes. E. coli, for example, carries its genetic instructions in a single, circular, double-stranded molecule of DNA that contains 4.6 million nucleotide pairs and 4300 protein-coding genes. (We focus on the genes that code for proteins because they are the best characterized, and their numbers are the most certain. We review how genes are counted in Chapter 9.) The simplest known bacterium contains only about 500 protein-coding genes, but most prokaryotes have genomes that contain at least 1 million nucleotide pairs and 1000–8000 protein-coding genes. With these few thousand genes, prokaryotes are able to thrive in even the most hostile environments on Earth. The compact genomes of typical bacteria are dwarfed by the genomes of typical eukaryotes. The human genome, for example, contains about 700 times more DNA than the E. coli genome, and the genome of an amoeba contains about 100 times more than ours (Figure 1–41). The rest of the E. coli BACTERIA Halobacterium sp. Figure 1−41 Organisms vary enormously ARCHAEA in the size of their genomes. Genome size malarial parasite amoeba is measured in nucleotide pairs of DNA per PROTOZOANS haploid genome; that is, per single copy yeast (S. cerevisiae) of the genome. (The body cells of sexually FUNGI reproducing organisms such as ourselves Arabidopsis wheat are generally diploid: they contain two PLANTS, ALGAE copies of the genome, one inherited from Caenorhabditis the mother, the other from the father.) NEMATODE WORMS Closely related organisms can vary widely Drosophila shrimp in the quantity of DNA in their genomes (as CRUSTACEANS, INSECTS indicated by the length of the green bars), zebrafish frog newt even though they contain similar numbers AMPHIBIANS, FISHES of functionally distinct genes; this is because human most of the DNA in large genomes does not MAMMALS, BIRDS, REPTILES code for protein, as discussed shortly. (Data from T.R. Gregory, 2008, Animal Genome 105 106 107 108 109 1010 1011 1012 Size Database: www.genomesize.com.) nucleotide pairs per haploid genome  Model Organisms 35 TABLE 1–2 SOME MODEL ORGANISMS AND THEIR GENOMES Organism Genome Size* Approximate Number (Nucleotide of Protein-coding Pairs) Genes Homo sapiens (human) 3200 × 106 19,000 Mus musculus (mouse) 2800 × 106 22,000 Drosophila melanogaster (fruit fly) 180 × 106 14,000 Arabidopsis thaliana (plant) 103 × 106 28,000 Caenorhabditis elegans (roundworm) 100 × 106 22,000 Saccharomyces cerevisiae (yeast) 12.5 × 106 6600 Escherichia coli (bacterium) 4.6 × 106 4300 *Genome size includes an estimate for the amount of highly repeated, noncoding DNA sequence, which does not appear in genome databases. model organisms we have described have genomes that fall somewhere between E. coli and human in terms of size. S. cerevisiae contains about 2.5 times as much DNA as E. coli; D. melanogaster has about 10 times more DNA than S. cerevisiae; and M. musculus has about 20 times more DNA than D. melanogaster (Table 1–2). In terms of gene numbers, however, the differences are not so great. We have only about five times as many protein-coding genes as E. coli, for example. Moreover, many of our genes—and the proteins they encode— fall into closely related family groups, such as the family of hemoglobins, which has nine closely related members in humans. Thus the number of fundamentally different proteins in a human is not very many times more than in the bacterium, and the number of human genes that have iden- tifiable counterparts in the bacterium is a significant fraction of the total. This high degree of “family resemblance” is striking when we compare the genome sequences of different organisms. When genes from different organisms have very similar nucleotide sequences, it is highly probable that they descended from a common ancestral gene. Such genes (and their protein products) are said to be homologous. Now that we have the complete genome sequences of many different organisms from all three domains of life—archaea, bacteria, and eukaryotes—we can search sys- tematically for homologies that span this enormous evolutionary divide. By taking stock of the common inheritance of all living things, scientists are attempting to trace life’s origins back to the earliest ancestral cells. We return to this topic in Chapter 9. Genomes Contain More Than Just Genes Although our view of genome sequences tends to be “gene-centric,” our genomes contain much more than just genes. The vast bulk of our DNA does not code for proteins or for functional RNA molecules. Instead, it includes a mixture of sequences that help regulate gene activity, plus sequences that seem to be dispensable. The large quantity of regulatory DNA contained in the genomes of eukaryotic multicellular organisms allows for enormous complexity and sophistication in the way different genes are brought into action at different times and places. Yet, in the end, the basic list of parts—the set of proteins that the cells can make, as specified by the DNA—is not much longer than the parts list of an auto- mobile, and many of those parts are common not only to all animals, but also to the entire living world. 36 CHAPTER 1 Cells: The Fundamental Units of Life That DNA can program the growth, development, and reproduction of living cells and complex organisms is truly amazing. In the rest of this book, we will try to explain what is known about how cells work—by examining their component parts, how these parts work together, and how the genome of each cell directs the manufacture of the parts the cell needs to function and to reproduce. ESSENTIAL CONCEPTS Cells are the fundamental units of life. All present-day cells are believed to have evolved from an ancestral cell that existed more than 3 billion years ago. All cells are enclosed by a plasma membrane, which separates the inside of the cell from its environment. All cells contain DNA as a store of genetic information and use it to guide the synthesis of RNA molecules and proteins. This molecular relationship underlies cells’ ability to self-replicate. Cells in a multicellular organism, though they all contain the same DNA, can be very different because they turn on different sets of genes according to their developmental history and to signals they receive from their environment. Animal and plant cells are typically 5–20 μm in diameter and can be seen with a light microscope, which also reveals some of their inter- nal components, including the larger organelles. The electron microscope reveals even the smallest organelles, but specimens require elaborate preparation and cannot be viewed while alive. Specific large molecules can be located in fixed or living cells by fluo- rescence microscopy. The simplest of present-day living cells are prokaryotes—bacteria and archaea: although they contain DNA, they lack a nucleus and most other organelles and probably resemble most closely the origi- nal ancestral cell. Different species of prokaryotes are diverse in their chemical capa- bilities and inhabit an amazingly wide range of habitats. Eukaryotic cells possess a nucleus and other organelles not found in prokaryotes. They probably evolved in a series of stages, including the acquisition of mitochondria by engulfment of aerobic bacteria and (for cells that carry out photosynthesis) the acquisition of chlo- roplasts by engulfment of photosynthetic bacteria. The nucleus contains the main genetic information of the eukaryotic organism, stored in very long DNA molecules. The cytoplasm of eukaryotic cells includes all of the cell’s contents outside the nucleus and contains a variety of membrane-enclosed organelles with specialized functions: mitochondria carry out the final oxidation of food molecules and produce ATP; the endoplasmic reticu- lum and the Golgi apparatus synthesize complex molecules for export from the cell and for insertion in cell membranes; lysosomes digest large molecules; in plant cells and other photosynthetic eukaryotes, chloroplasts perform photosynthesis. Outside the membrane-enclosed organelles in the cytoplasm is the cytosol, a highly concentrated mixture of large and small molecules that carry out many essential biochemical processes. The cytoskeleton is composed of protein filaments that extend throughout the cytoplasm and are responsible for cell shape and movement and for the transport of organelles and large molecular complexes from one intracellular location to another. CHAPTER FOUR 4 Protein Structure and Function When we look at a cell in a microscope or analyze its electrical or bio- THE SHAPE AND STRUCTURE chemical activity, we are, in essence, observing the handiwork of proteins. OF PROTEINS Proteins are the main building blocks from which cells are assembled, and they constitute most of the cell’s dry mass. In addition to provid- ing the cell with shape and structure, proteins also execute nearly all its HOW PROTEINS WORK myriad functions. Enzymes promote intracellular chemical reactions by providing intricate molecular surfaces contoured with particular bumps HOW PROTEINS ARE and crevices that can cradle or exclude specific molecules. Transporters and channels embedded in the plasma membrane control the passage CONTROLLED of nutrients and other small molecules into and out of the cell. Other proteins carry messages from one cell to another, or act as signal inte- HOW PROTEINS ARE STUDIED grators that relay information from the plasma membrane to the nucleus of individual cells. Some proteins act as motors that propel organelles through the cytosol, and others function as components of tiny molecu- lar machines with precisely calibrated moving parts. Specialized proteins also act as antibodies, toxins, hormones, antifreeze molecules, elastic fibers, or luminescence generators. To understand how muscles contract, how nerves conduct electricity, how embryos develop, or how our bodies function, we must first understand how proteins operate. The multiplicity of functions carried out by these remarkable macromol- ecules, a few of which are represented in Panel 4−1, p. 118, arises from the huge number of different shapes proteins adopt. We therefore begin our description of proteins by discussing their three-dimensional struc- tures and the properties that these structures confer. We next look at how proteins work: how enzymes catalyze chemical reactions, how some proteins act as molecular switches, and how others generate orderly movement. We then examine how cells control the activity and location 118 PANEL 4–1 A FEW EXAMPLES OF SOME GENERAL PROTEIN FUNCTIONS ENZYMES STRUCTURAL PROTEINS TRANSPORT PROTEINS function: Catalyze covalent bond breakage function: Provide mechanical support to function: Carry small molecules or ions or formation cells and tissues examples: In the bloodstream, serum albumin examples: Living cells contain thousands of carries lipids, hemoglobin carries oxygen, and different enzymes, each of which catalyzes transferrin carries iron. Many proteins embedded (speeds up) one particular reaction. Examples examples: Outside cells, collagen and elastin in cell membranes transport ions or small include: alcohol dehydrogenase—makes the are common constituents of extracellular molecules across the membrane. For example, the alcohol in wine; pepsin—degrades dietary matrix and form fibers in tendons and bacterial protein bacteriorhodopsin is a proteins in the stomach; ribulose ligaments. Inside cells, tubulin forms long, stiff light-activated proton pump that transports H+ bisphosphate carboxylase—helps convert microtubules, and actin forms filaments that ions out of the cell; glucose transporters shuttle carbon dioxide into sugars in plants; DNA underlie and support the plasma membrane; glucose into and out of cells; and a Ca2+ pump polymerase—copies DNA; protein kinase — keratin forms fibers that reinforce epithelial clears Ca2+ from a muscle cell’s cytosol after the adds a phosphate group to a protein cells and is the major protein in hair and horn. ions have triggered a contraction. molecule. MOTOR PROTEINS STORAGE PROTEINS SIGNAL PROTEINS function: Generate movement in cells and function: Store amino acids or ions function: Carry extracellular signals from tissues cell to cell examples: Many of the hormones and growth factors that coordinate physiological functions in animals are proteins. Insulin, for example, is a small protein that controls glucose levels in the blood; netrin attracts growing nerve cell examples: Myosin in skeletal muscle cells examples: Iron is stored in the liver by binding axons to specific locations in the developing provides the motive force for humans to to the small protein ferritin; ovalbumin in egg spinal cord; nerve growth factor (NGF) move; kinesin interacts with microtubules to white is used as a source of amino acids for stimulates some types of nerve cells to grow move organelles around the cell; dynein the developing bird embryo; casein in milk is a axons; epidermal growth factor (EGF) enables eukaryotic cilia and flagella to beat. source of amino acids for baby mammals. stimulates the growth and division of epithelial cells. RECEPTOR PROTEINS TRANSCRIPTION REGULATORS SPECIAL-PURPOSE PROTEINS function: Detect signals and transmit them function: Bind to DNA to switch genes on function: Highly variable to the cell's response machinery or off examples: Organisms make many proteins with highly specialized properties. These molecules examples: Rhodopsin in the retina detects illustrate the amazing range of functions that light; the acetylcholine receptor in the proteins can perform. The antifreeze proteins of membrane of a muscle cell is activated by Arctic and Antarctic fishes protect their blood acetylcholine released from a nerve ending; examples: The Lac repressor in bacteria against freezing; green fluorescent protein from the insulin receptor allows a cell to respond to silences the genes for the enzymes that jellyfish emits a green light; monellin, a protein the hormone insulin by taking up glucose; the degrade the sugar lactose; many different found in an African plant, has an intensely sweet adrenergic receptor on heart muscle increases DNA-binding proteins act as genetic switches taste; mussels and other marine organisms secrete the rate of the heartbeat when it binds to to control development in multicellular glue proteins that attach them firmly to rocks, epinephrine secreted by the adrenal gland. organisms, including humans. even when immersed in seawater.  The Shape and Structure of Proteins 119 of the proteins they contain. Finally, we present a brief description of the techniques that biologists use to work with proteins, including methods for purifying them—from tissues or cultured cells—and for determining their structures. THE SHAPE AND STRUCTURE OF PROTEINS From a chemical point of view, proteins are by far the most structurally complex and functionally sophisticated molecules known. This is per- haps not surprising, considering that the structure and activity of each protein has developed and been fine-tuned over billions of years of evo- lution. We start by considering how the position of each amino acid in the long string of amino acids that forms a protein determines its three- dimensional conformation, a shape that is stabilized by noncovalent interactions between different parts of the molecule. Understanding the structure of a protein at the atomic level allows us to see how the precise shape of the protein determines its function. The Shape of a Protein Is Specified by Its Amino Acid Sequence Proteins, as you may recall from Chapter 2, are assembled mainly from a set of 20 different amino acids, each with different chemical properties. A protein molecule is made from a long chain of these amino acids, held together by covalent peptide bonds (Figure 4–1). Proteins are therefore referred to as polypeptides, or polypeptide chains. In each type of pro- tein, the amino acids are present in a unique order, called the amino acid sequence, which is exactly the same from one molecule of that protein to the next. One molecule of human insulin, for example, should have the same amino acid sequence as every other molecule of human insulin. Many thousands of different proteins have been identified, each with its own distinct amino acid sequence. Each polypeptide chain consists of a backbone that is adorned with a variety of chemical side chains. The polypeptide backbone is formed from a repeating sequence of the core atoms (–N–C–C–) found in every amino group carboxyl group + + – – glycine alanine PEPTIDE BOND FORMATION WITH REMOVAL OF WATER water Figure 4–1 Amino acids are linked together by peptide bonds. A covalent peptide bond forms when the carbon atom of the carboxyl group of one amino acid (such as glycine) shares electrons with the nitrogen atom from the amino group of a second amino acid + (such as alanine). Because a molecule of water is eliminated, peptide bond formation is classified as a condensation reaction (see – Figure 2−31). In this diagram, carbon atoms are black, nitrogen blue, oxygen red, and peptide bond in glycylalanine hydrogen white. 120 CHAPTER 4 Protein Structure and Function Figure 4–2 A protein is made of OH amino acids linked together into a O O polypeptide chain. The amino acids are linked by peptide bonds (see C Figure 4–1) to form a polypeptide polypeptide backbone side chains backbone of repeating structure (gray CH2 CH2 boxes), from which the side chain H H O H H O O of each amino acid projects. The amino terminus + carboxyl terminus sequence of these chemically distinct (N-terminus) H N C C N C C N C C N C C (C-terminus) side chains—which can be nonpolar O H H H O H H (green), polar uncharged (yellow), positively charged (red ), or negatively CH2 CH2 charged (blue)—gives each protein its peptide peptide bond C H bonds CH distinct, individual properties. A small polypeptide of just four amino acids HN C H3C CH3 is shown here. Proteins are typically HC N made up of chains of several hundred side chains amino acids, whose sequence is always H+ presented starting with the N-terminus and read from left to right. Histidine Aspartic acid Leucine Tyrosine (His) (Asp) (Leu) (Tyr) amino acid (Figure 4–2). Because the two ends of each amino acid are chemically different—one sports an amino group (NH3+, also written NH2) and the other a carboxyl group (COO–, also written COOH)—each poly- peptide chain has a directionality: the end carrying the amino group is called the amino terminus, or N-terminus, and the end carrying the free carboxyl group is the carboxyl terminus, or C-terminus. ECB5 e4.02/4.02 Projecting from the polypeptide backbone are the amino acid side chains—the part of the amino acid that is not involved in forming peptide bonds (see Figure 4–2). The side chains give each amino acid its unique properties: some are nonpolar and hydrophobic (“water-fearing”), some are negatively or positively charged, some can be chemically reactive, and so on. The atomic formula for each of the 20 amino acids in proteins is presented in Panel 2–6 (pp. 76–77), and a brief list of the 20 common amino acids, with their abbreviations, is provided in Figure 4–3. Long polypeptide chains are very flexible, as many of the covalent bonds that link the carbon atoms in the polypeptide backbone allow free rota- tion of the atoms they join. Thus, proteins can in principle fold in an AMINO ACID SIDE CHAIN AMINO ACID SIDE CHAIN Aspartic acid Asp D negatively charged Alanine Ala A nonpolar Glutamic acid Glu E negatively charged Glycine Gly G nonpolar Arginine Arg R positively charged Valine Val V nonpolar Lysine Lys K positively charged Leucine Leu L nonpolar Histidine His H positively charged Isoleucine Ile I nonpolar Asparagine Asn N uncharged polar Proline Pro P nonpolar Glutamine Gln Q uncharged polar Phenylalanine Phe F nonpolar Serine Ser S uncharged polar Methionine Met M nonpolar Threonine Thr T uncharged polar Tryptophan Trp W nonpolar Tyrosine Tyr Y uncharged polar Cysteine Cys C nonpolar POLAR AMINO ACIDS NONPOLAR AMINO ACIDS Figure 4–3 Twenty different amino acids are commonly found in proteins. Both three-letter and one-letter abbreviations are given, as well as the character of the side chain. There are equal numbers of polar (hydrophilic) and nonpolar (hydrophobic) side chains, and half of the polar side chains are charged at neutral pH in an aqueous solution. The structures of all of these amino acids are shown in Panel 2−6, pp. 76−77. ECB5 e4.03-4.03  The Shape and Structure of Proteins 121 glutamic acid H O N C C electrostatic H attractions CH2 + R CH2 C C H hydrogen bond H O O H C N H H O C O N H + C H H CH2 C R N CH2 van der Waals attractions C CH2 R O CH2 H C O H CH3 CH3 C C C C H N CH3 CH3 valine O H H HN Figure 4–4 Three types of noncovalent C CH3 lysine N C bonds help proteins fold. Although a H C H single one of any of these bonds is quite C N C O H H weak, many of them together can create a O strong bonding arrangement that stabilizes valine alanine a particular three-dimensional structure, as in the small polypeptide shown in the center. R is often used as a general designation for an amino acid side chain. Protein folding is also aided by hydrophobic forces, as shown in Figure 4–5. enormous number of ways. The shape of each of these folded chains, however, is constrained by many sets of weak noncovalent bonds that ECB5 e4.04/4.04 form within proteins. These bonds involve atoms in the polypeptide backbone, as well as atoms within the amino acid side chains. The non- covalent bonds that help proteins fold up and maintain their shape include hydrogen bonds, electrostatic attractions, and van der Waals attractions, which are described in Chapter 2 (see Panel 2–3, pp. 70–71). Because a noncovalent bond is much weaker than a covalent bond, it takes many noncovalent bonds to hold two regions of a polypeptide chain tightly together. The stability of each folded shape is largely determined by the combined strength of large numbers of noncovalent bonds (Figure 4–4). A fourth weak interaction, the hydrophobic force, also has a central role in determining the shape of a protein. In an aqueous environment, hydro- phobic molecules, including the nonpolar side chains of particular amino acids, tend to be forced together to minimize their disruptive effect on the hydrogen-bonded network of the surrounding water molecules (see Panel 2−3, pp. 70–71). Therefore, an important factor governing the fold- ing of any protein is the distribution of its polar and nonpolar amino acids. The nonpolar (hydrophobic) side chains—which belong to amino acids such as phenylalanine, leucine, valine, and tryptophan (see Figure 4–3)—tend to cluster in the interior of the folded protein (just as hydro- phobic oil droplets coalesce to form one large drop). Tucked away inside the folded protein, hydrophobic side chains can avoid contact with the aqueous environment that surrounds them inside a cell. In contrast, polar side chains—such as those belonging to arginine, glutamine, and histi- dine—tend to arrange themselves near the outside of the folded protein, where they can form hydrogen bonds with water and with other polar molecules (Figure 4–5). When polar amino acids are buried within the protein, they are usually hydrogen-bonded to other polar amino acids or to the polypeptide backbone (Figure 4–6). 122 CHAPTER 4 Protein Structure and Function Figure 4–5 Hydrophobic forces help unfolded polypeptide proteins fold into compact conformations. In a folded protein, polar amino acid side chains tend to be displayed on the surface, where they can interact with water; nonpolar amino acid side chains are buried on the inside to form a tightly packed hydrophobic core of atoms that are hidden from water. nonpolar polar polypeptide side chains side chains backbone polar side chains nonpolar side chains can form hydrogen are packed into bonds to water hydrophobic core region folded conformation in aqueous environment Proteins Fold into a Conformation of Lowest Energy Each type of protein has a particular three-dimensional structure, which is determined by the order of m3.05/4.05 ECB5 the amino acids in its polypeptide chain. The final folded structure, or conformation, adopted by any polypeptide chain is determined by energetic considerations: a protein generally folds into the shape in which its free energy (G) is minimized. The folding pro- cess is thus energetically favorable, as it releases heat and increases the disorder of the universe (see Panel 3−1, pp. 94–95). Figure 4–6 Hydrogen bonds within a 42 protein molecule help stabilize its folded shape. Large numbers of hydrogen bonds form between adjacent regions of a folded polypeptide chain. The structure shown is a portion of the enzyme lysozyme, between amino acids 42 and 63. Hydrogen bonds between two atoms in the polypeptide backbone are shown in red ; those between the backbone and a side chain are shown in yellow ; and those between atoms of two side chains are shown in blue. Note that the same amino acid side chain can make 63 multiple hydrogen bonds (red arrow). In this diagram, nitrogen atoms are blue, oxygen atoms are red, and carbon atoms are gray; backbone to backbone backbone to side chain side chain to side chain hydrogen atoms are not shown. (After C.K. Mathews, K.E. van Holde, and K.G. hydrogen bond between hydrogen bond between hydrogen bond between Ahern, Biochemistry, 3rd ed. San Francisco: atoms of two peptide atoms of a peptide bond atoms of two amino bonds and an amino acid side chain acid side chains Benjamin Cummings, 2000.)  The Shape and Structure of Proteins 123 Figure 4–7 Denatured proteins can EXPOSE TO A HIGH often recover their natural shapes. This CONCENTRATION REMOVE type of experiment demonstrates that the OF UREA UREA conformation of a protein is determined solely by its amino acid sequence. Renaturation requires the correct conditions purified protein protein refolds into its isolated from cells original conformation and works best for small proteins. denatured protein Protein folding has been studied in the laboratory using highly purified proteins. A protein can be unfolded, or denatured, by treatment with sol- QUESTION 4–1 vents that disrupt the noncovalent interactions holding the folded chain together. This treatment converts the protein into a flexible polypeptide Urea, used in the experiment shown chain that has lost its natural shape. Under the right conditions, when the in Figure 4−7, is a molecule that denaturing solvent is removed, the protein often refolds spontaneously disrupts the hydrogen-bonded ECB5process 04.07 called renaturation (Figure 4–7). network of water molecules. Why into its original conformation—a might high concentrations of urea The fact that a denatured protein can, on its own, refold into the cor- unfold proteins? The structure of rect conformation indicates that all the information necessary to specify urea is shown here. the three-dimensional shape of a protein is contained in its amino acid sequence. O Although a protein chain can fold into its correct conformation without C outside help, protein folding in a living cell is generally assisted by a large H2N NH2 set of special proteins called chaperone proteins. Some of these chaper- ones bind to partly folded chains and help them to fold along the most energetically favorable pathway (Figure 4–8). Others form “isolation chambers” in which single polypeptide chains can fold without the risk of forming aggregates in the crowded conditions of the cytoplasm (Figure ECB4 Q4.01/Q4.01 4–9). In either case, the final three-dimensional shape of the protein is still specified by its amino acid sequence; chaperones merely make the folding process more efficient and reliable. Each protein normally folds into a single, stable conformation. This con- formation, however, often changes slightly when the protein interacts with other molecules in the cell. Such changes in shape are crucial to the function of the protein, as we discuss later. newly synthesized, partially folded protein chaperone proteins incorrectly folded correctly folded protein protein Figure 4–8 Chaperone proteins can guide the folding of a newly synthesized polypeptide chain. The chaperones bind to newly synthesized or partially folded chains and help them to fold along the most energetically favorable pathway. The function of these chaperones requires ATP binding and hydrolysis. ECB5 04.08 124 CHAPTER 4 Protein Structure and Function newly synthesized, chamber partially folded proteins cap chaperone one polypeptide isolated correctly folded protein chain is sequestered polypeptide protein is released by the chaperone chain folds when cap correctly dissociates Figure 4–9 Some chaperone proteins act as isolation chambers that help a polypeptide fold. In this case, the barrel of the chaperone provides an enclosed chamber in which a newly synthesized polypeptide chain can fold without the risk of aggregating with other polypeptidesECB5 04.09 in the crowded conditions of the cytoplasm. This system also requires an input of energy from ATP hydrolysis, mainly for the association and subsequent dissociation of the cap that closes off the chamber. Proteins Come in a Wide Variety of Complicated Shapes Proteins are the most structurally diverse macromolecules in the cell. Although they range in size from about 30 amino acids to more than 10,000, the vast majority are between 50 and 2000 amino acids long. Proteins can be globular or fibrous, and they can form filaments, sheets, rings, or spheres (Figure 4−10). We will encounter many of these struc- tures throughout the book. To date, the structures of about 100,000 different proteins have been determined (using techniques we discuss later in the chapter). Most pro- teins have a three-dimensional conformation so intricate and irregular that their structure would require the rest of the chapter to describe in detail. But we can get some sense of the intricacies of polypeptide struc- ture by looking at the conformation of a relatively small protein, such as the bacterial transport protein HPr. This small protein, only 88 amino acids long, facilitates the transport of sugar into bacterial cells. In Figure 4−11, we present HPr’s three- dimensional structure in four different ways, each of which emphasizes different features of the protein. The backbone model (see Figure 4−11A) shows the overall organization of the polypeptide chain and provides a straightforward way to compare the structures of related proteins. The ribbon model (see Figure 4−11B) shows the polypeptide backbone in a way that emphasizes its most conspicuous folding patterns, which we describe in detail shortly. The wire model (see Figure 4−11C) includes the positions of all the amino acid side chains; this view is especially useful for predicting which amino acids might be involved in the protein’s activ- ity. Finally, the space-filling model (see Figure 4−11D) provides a contour map of the protein surface, which reveals which amino acids are exposed on the surface and shows how the protein might look to a small molecule such as water or to another macromolecule in the cell. The structures of larger proteins—or of multiprotein complexes—are even more complicated. To visualize such detailed and intricate structures, scientists have developed various computer-based tools to empha- size different features of a protein, only some of which are depicted in Figure 4–11. All of these images can be displayed on a computer screen and readily rotated and magnified to view all aspects of the structure (Movie 4.1). When the three-dimensional structures of many different protein mol- ecules are compared, it becomes clear that, although the overall 126 CHAPTER 4 Protein Structure and Function (A) backbone model Figure 4−11 Protein conformation can be represented in a variety of ways. Shown here is the structure of the small bacterial transport protein HPr. The images are colored to make it easier to trace the path of the polypeptide chain. In these models, the region of polypeptide chain carrying the protein’s N-terminus is purple and that near its C-terminus is red. conformation of each protein is unique, some regular folding patterns can be detected, as we discuss next. The α Helix and the β Sheet Are Common Folding (B) ribbon model Patterns More than 60 years ago, scientists studying hair and silk discovered two regular folding patterns that are present in many different proteins. The first to be discovered, called the α helix, was found in the protein α-keratin, which is abundant in skin and its derivatives—such as hair, nails, and horns. Within a year of that discovery, a second folded structure, called a β sheet, was found in the protein fibroin, the major constituent of silk. (Biologists often use Greek letters to name their discoveries, with the first example receiving the designation α, the second β, and so on.) These two folding patterns are particularly common because they result from hydrogen bonds that form between the N–H and C=O groups in (C) wire model the polypeptide backbone (see Figure 4−6). Because the amino acid side chains are not involved in forming these hydrogen bonds, α helices and β sheets can be generated by many different amino acid sequences. In each case, the protein chain adopts a regular, repeating form. These structural features, and the shorthand cartoon symbols that are often used to repre- sent them in models of protein structures, are presented in Figures 4−12 and 4−13. α helix amino acid R side chain (D) space-filling model R R oxygen R hydrogen bond 0.54 nm R carbon R hydrogen R carbon R nitrogen nitrogen R (A) (B) (C) Figure 4−12 Some polypeptide chains fold into an orderly repeating form known as an α helix. (A) In an α helix, the N–H of every peptide bond is hydrogen- bonded to the C=O of a neighboring peptide bond located four amino acids away in the same chain. All of the atoms in the polypeptide backbone are shown; the amino acid side chains are denoted by R. (B) The same polypeptide, showing only the carbon (black and gray) and nitrogen (blue) atoms. (C) Cartoon symbol used to represent an α helix in ribbon models of proteins (see Figure 4−11B). ECB5 e4.13/4.13  The Shape and Structure of Proteins 127 β sheet Figure 4−13 Some polypeptide chains peptide fold into an orderly pattern called a (A) bond carbon β sheet. (A) In a β sheet, several segments R oxygen R nitrogen (strands) of an individual polypeptide chain are held together by hydrogen-bonding between peptide bonds in adjacent R R strands. The amino acid side chains in hydrogen R hydrogen each strand project alternately above R R R bond and below the plane of the sheet. In the R example shown, the adjacent chains run in R R R opposite directions, forming an antiparallel β sheet. All of the atoms in the polypeptide backbone are shown; the amino acid side carbon R chains are denoted by R. (B) The same R R polypeptide, showing only the carbon amino acid (black and gray) and nitrogen (blue) atoms. side chain (C) Cartoon symbol used to represent (B) β sheets in ribbon models of proteins (see Figure 4−11B).