Proteins are designed to bind every conceivable molecule—from simple ions to large complex molecules like fats, sugars, nucleic acids, and other proteins. They catalyze an extraordinary range of chemical reactions, provide structural rigidity to the cell, control flow of material through membranes, regulate the concentrations of metabolites, act as sensors and switches, cause motion, and control gene function. The three-dimensional structures of proteins have evolved to carry out these functions efficiently and under precise control. The spatial organization of proteins, their shape in three dimensions, is a key to understanding how they work.
One of the major areas of biological research today is how proteins, constructed from only 20 different amino acids, carry out the incredible array of diverse tasks that they do. Unlike the intricate branched structure of carbohydrates, proteins are single, unbranched chains of amino acid monomers. The unique shape of proteins arises from noncovalent interactions between regions in the linear sequence of amino acids. Only when a protein is in its correct three-dimensional structure, or conformation, is it able to function efficiently. A key concept in understanding how proteins work is that function is derived from three-dimensional structure, and three-dimensional structure is specified by amino acid sequence.
The α carbon atom (green) of each amino acid is bonded to four different chemical groups and thus is asymmetric. The side chain, or R group (red), is unique to each amino acid. The diversity of natural proteins reflects different linear combinations of the 20 naturally occurring amino acids. The short peptide shown here, containing only four amino acids, has 204, or 160,000, possible sequences.
). All 20 different amino
acids have this same general structure, but their side-chain groups vary in size, shape,
charge, hydrophobicity, and reactivity.
The side chain determines the characteristic properties of each amino acid. Shown are the zwitterion forms, which exist at the pH of the cytosol. In parentheses are the three-letter and one-letter abbreviations for each amino acid.
). Amino acids with polar side groups
tend to be on the surface of proteins; by interacting with water, they make proteins soluble in
aqueous solutions. In contrast, amino acids with nonpolar side groups avoid water and aggregate to form the waterinsoluble core of
proteins. The polarity of amino acid side chains thus is one of the forces responsible for
shaping the final three-dimensional structure of proteins.
Hydrophilic, or water-soluble, amino acids have ionized or polar side chains. At neutral pH, arginine and lysine are positively charged; aspartic acid and glutamic acid are negatively charged and exist as aspartate and glutamate. These four amino acids are the prime contributors to the overall charge of a protein. A fifth amino acid, histidine, has an imidazole side chain, which has a pKa of 6.8, the pH of the cytoplasm. As a result, small shifts of cellular pH will change the charge of histidine side chains:
The activities of many proteins are modulated by pH through protonation of histidine side chains. Asparagine and glutamine are uncharged but have polar amide groups with extensive hydrogen-bonding capacities. Similarly, serine and threonine are uncharged but have polar hydroxyl groups, which also participate in hydrogen bonds with other polar molecules. Because the charged and polar amino acids are hydrophilic, they are usually found at the surface of a water-soluble protein, where they not only contribute to the solubility of the protein in water but also form binding sites for charged molecules.
Hydrophobic amino acids have aliphatic side chains, which are insoluble or only slightly soluble in water. The side chains of alanine, valine, leucine, isoleucine, and methionine consist entirely of hydrocarbons, except for the sulfur atom in methionine, and all are nonpolar. Phenylalanine, tyrosine, and tryptophan have large bulky aromatic side groups. As explained in Chapter 2, hydrophobic molecules avoid water by coalescing into an oily or waxy droplet. The same forces cause hydrophobic amino acids to pack in the interior of proteins, away from the aqueous environment. Later in this chapter, we will see in detail how hydrophobic residues line the surface of membrane proteins that reside in the hydrophobic environment of the lipid bilayer.
Lastly, cysteine, glycine, and proline exhibit special roles in proteins because of the unique properties of their side chains. The side chain of cysteine contains a reactive sulfhydryl group (—SH), which can oxidize to form a disulfide bond (—S—S—) to a second cysteine:
Regions within a protein chain or in separate chains sometimes are cross-linked covalently through disulfide bonds. Although disulfide bonds are rare in intracellular proteins, they are commonly found in extracellular proteins, where they help maintain the native, folded structure. The smallest amino acid, glycine, has a single hydrogen atom as its R group. Its small size allows it to fit into tight spaces. Unlike any of the other common amino acids, proline has a cyclic ring that is produced by formation of a covalent bond between its R group and the amino group on Cα. Proline is very rigid, and its presence creates a fixed kink in a protein chain. Proline and glycine are sometimes found at points on a protein’s surface where the chain loops back into the protein.
The 6225 known and predicted proteins encoded by the yeast genome have an average molecular weight (MW) of 52,728 and contain, on average, 466 amino acid residues. Assuming that these average values represent a “typical” eukaryotic protein, then the average molecular weight of amino acids is 113, taking their average relative abundance in proteins into account. This is a useful number to remember, as we can use it to estimate the number of residues from the molecular weight of a protein or vice versa. Some amino acids are more abundant in proteins than other amino acids. Cysteine, tryptophan, and methionine are rare amino acids; together they constitute approximately 5 percent of the amino acids in a protein. Four amino acids—leucine, serine, lysine, and glutamic acid—are the most abundant amino acids, totaling 32 percent of all the amino acid residues in a typical protein. However, the amino acid composition of proteins can vary widely from these values. For example, as discussed in later sections, proteins that reside in the lipid bilayer are enriched in hydrophobic amino acids.
(a) A condensation reaction between two amino acids forms the peptide bond, which links all the adjacent residues in a protein chain. (b) Side-chain groups (R) extend from the backbone of a protein chain, in which the amino N, α carbon, carbonyl carbon sequence is repeated throughout.
). The repeated amide N,
Cα, and carbonyl C atoms of each amino acid residue form the backbone of
a protein molecule from which the various side-chain groups project. As a consequence of the
peptide linkage, the backbone has polarity, since all the amino groups lie to the same side of
the Cα atoms. This leaves at opposite ends of the chain a free (unlinked)
amino group (the N-terminus) and a free carboxyl group (the C-terminus). A protein chain is
conventionally depicted with its N-terminal amino acid on the left and its C-terminal amino
acid on the right (Figure 3-3b).
Many terms are used to denote the chains formed by polymerization of amino acids. A short chain of amino acids linked by peptide bonds and having a defined sequence is a peptide; longer peptides are referred to as polypeptides. Peptides generally contain fewer than 20–30 amino acid residues, whereas polypeptides contain as many as 4000 residues. We reserve the term protein for a polypeptide (or a complex of polypeptides) that has a threedimensional structure. It is implied that proteins and peptides represent natural products of a cell.
The size of a protein or a polypeptide is reported as its mass in daltons (a dalton is 1 atomic mass unit) or as its molecular weight (a dimensionless number). For example, a 10,000-MW protein has a mass of 10,000 daltons (Da), or 10 kilodaltons (kDa). In the last section of this chapter, we will discuss different methods for measuring the sizes and other physical characteristics of proteins.
(a) Primary structure is illustrated by the amino acid sequence of residues 68 –195 of HA1. This region is used by influenza virus to bind to animal cells. The one-letter amino acid code is used. Secondary structure is represented diagrammatically beneath the sequence, showing regions of the polypeptide chain that are folded into α helices (light blue cylinders), β strands (light green arrows), and random coils (white strands). (b) Tertiary structure constitutes the folding of the helices and strands in each HA subunit into a compact structure that is 13.5 nm long and divided into two domains. The membrane-distal domain is folded into a globular conformation. The blue and green segments in this domain correspond to the sequence shown in part (a). The proximal domain, which lies adjacent to the viral membrane, has a stemlike conformation due to alignment of two long helices of HA2 (dark blue) with β strands in HA1. Short turns and longer loops, which usually lie at the surface of the molecule, connect the helices and strands in a given chain. (c) The quaternary structure comprises the three subunits of HA; the structure is stabilized by lateral interactions among the long helices (dark blue) in the subunit stems, forming a triple-stranded coiled-coil stalk. Each of the distal globular domains in trimeric hemagglutinin has a site (red) for binding sialic acid molecules on the surface of target cells. Like many membrane proteins, HA has several covalently bound carbohydrate (CHO) chains.
, which depicts the structure of hemagglutinin, a surface protein on the influenza
virus. This protein binds to the surface of animal cells, including human cells, and is
responsible for the infectivity of the flu virus.
The primary structure of a protein is the linear arrangement, or sequence, of amino acid residues that constitute the polypeptide chain.
Secondary structure refers to the localized organization of parts of a polypeptide chain, which can assume several different spatial arrangements. A single polypeptide may exhibit all types of secondary structure. Without any stabilizing interactions, a polypeptide assumes a random-coil structure. However, when stabilizing hydrogen bonds form between certain residues, the backbone folds periodically into one of two geometric arrangements: an α helix, which is a spiral, rodlike structure, or a β sheet, a planar structure composed of alignments of two or more β strands, which are relatively short, fully extended segments of the backbone. Finally, U-shaped four-residue segments stabilized by hydrogen bonds between their arms are called turns. They are located at the surfaces of proteins and redirect the polypeptide chain toward the interior. (These structures will be discussed in greater detail later.)
Tertiary structure, the next-higher level of structure, refers to the overall conformation of a polypeptide chain, that is, the three-dimensional arrangement of all the amino acids residues. In contrast to secondary structure, which is stabilized by hydrogen bonds, tertiary structure is stabilized by hydrophobic interactions between the nonpolar side chains and, in some proteins, by disulfide bonds. These stabilizing forces hold the α helices, β strands, turns, and random coils in a compact internal scaffold. Thus, a protein’s size and shape is dependent not only on its sequence but also on the number, size, and arrangement of its secondary structures. For proteins that consist of a single polypeptide chain, monomeric proteins, tertiary structure is the highest level of organization.
Multimeric proteins contain two or more polypeptide chains, or subunits, held together by noncovalent bonds. Quaternary structure describes the number (stoichiometry) and relative positions of the subunits in a multimeric protein. Hemagglutinin is a trimer of three identical subunits; other multimeric proteins can be composed of any number of identical or different subunits.
In a fashion similar to the hierarchy of structures that make up a protein, proteins themselves are part of a hierarchy of cellular structures. Proteins can associate into larger structures termed macromolecular assemblies. Examples of such macromolecular assemblies include the protein coat of a virus, a bundle of actin filaments, the nuclear pore complex, and other large submicroscopic objects. Macromolecular assemblies in turn combine with other cell biopolymers like lipids, carbohydrates, and nucleic acids to form complex cell organelles.
Guanosine diphosphate, the substrate that is bound, is shown as a blue space-filling figure in parts (a)–(d). (a) The Cαtrace of Ras, which highlights the course of the backbone. Evident from this view is how the polypeptide is packed into the smallest possible volume. (b) Ball-and-stick model of Ras showing the location of all atoms. (c) A schematic diagram of Ras showing how β strands (arrows) and α helices (cylinders) are organized in the protein. Note the turns and loops connecting pairs of helices and strands. (d) The water-accessible surface of Ras. Painted on the surface are regions of positive charge (blue) and negative charge (red). Here we see that the surface of a protein is not smooth but has lumps, bumps, and crevices. The molecular basis for specific binding interactions lies in the uneven distribution of charge over the surface of the protein. [Adapted from L. Tong et al., 1991, J. Mol. Biol.217:503; courtesy of S. Choe.]
); the most complex model shows
the location of every atom (Figure 3-5b; see also Figure 2-1a). The former shows the overall organization of
the polypeptide chain without consideration of the amino acid side chains; the latter details
the interactions among atoms that form the backbone and that stabilize the protein’s
conformation. Even though both views are useful, the elements of secondary structure are not
easily discerned in them.
). This type of representation emphasizes the organization of the
secondary structure of a protein, and various combinations of secondary structures are easily
seen.
). This view represents a
protein as seen by another molecule.In an average protein, 60 percent of the polypeptide chain exists as two regular secondary structures, α helices and β sheets; the remainder of the molecule is in random coils and turns. Thus, α helices and β sheets are the major internal supportive elements in proteins. In this section, we explore the forces that favor formation of secondary structures. In later sections, we examine how these structures can pack into larger arrays.
The polypeptide backbone is folded into a spiral that is held in place by hydrogen bonds (black dots) between backbone oxygen atoms and hydrogen atoms. Note that all the hydrogen bonds have the same polarity. The outer surface of the helix is covered by the side-chain R groups.
). The
stable arrangement of amino acids in the α helix holds the backbone as a rodlike
cylinder from which the side chains point outward. The hydrophobic or hydrophilic quality of
the helix is determined entirely by the side chains, because the polar groups of the peptide
backbone are already involved in hydrogen bonding in the helix and thus are unable to affect
its hydrophobicity or hydrophilicity.
The five chains of cartilage oligomeric matrix protein associate into a coiled-coil fibrous domain through amphipathic α helices. Seen in cross section through a part of the domain, the hy-drophobic residues (gray) face the interior, and the hydrophilic residues (yellow) line the surface. This arrangement of hydropho-bic and hydrophilic residues is typical of proteins in an aqueous environment. [Courtesy of V. Malashkevich.]
). One way of visualizing this arrangement is to look down the
center of an α helix and then project the amino acid residues onto the plane of the
paper. The residues will appear as a wheel, and in the case of an amphipathic helix, the
hydrophobic residues all lie on one side of the wheel and the hydrophilic ones on the other
side.
Amphipathic α helices are important structural elements in fibrous proteins found in a watery environment. In a coiled-coil region of a protein, the hydrophobic surface of the α helix faces inward to form the hydrophobic core, and the hydrophilic surfaces face outward toward the surrounding fluid. This same orientation of surfaces is also found in most globular proteins. A crucial difference is that the hydrophobic interaction could be with a β strand, random coil, or another α helix. As we discuss later, amphipathic β strands line the walls of an ion channel in the cell membrane.
(a) A simple two-stranded β sheet with antiparallel β strands. A sheet is stabilized by hydrogen bonds (black dots) between the β strands. The planarity of the peptide bond forces a β sheet to be pleated; hence, this structure is also called a β pleated sheet, or simply a pleated sheet. (b) Side view of a β sheet showing how the R groups protrude above and below the plane of the sheet. (c) Model of binding site in class I MHC (major histocompatibility complex) molecules, which are involved in graft rejection. A sheet comprising eight antiparallel β strands (green) forms the bottom of the binding cleft, which is lined by a pair of α helices (blue). A disulfide bond is shown as two connected yellow spheres. The MHC binding cleft is large enough to bind a peptide 8–10 residues long. [Part (b) adapted from C. Branden and J. Tooze, 1991, Introduction to Protein Structure, Garland.]
). Like α helices, β
strands have a polarity defined by the orientation of the peptide bond. Therefore, in a
pleated sheet, adjacent β strands can be oriented antiparallel or parallel with
respect to each other. In both arrangements of the backbone, the side chains project from both
faces of the sheet (Figure 3-8b).
). In many structural proteins, multiple layers of pleated
sheets provide toughness. Silk fibers, for example, consist almost entirely of stacks of
antiparallel β sheets. The fibers are flexible because the stacks of β
sheets can slip over one another. However, they are also resistant to breakage because the
peptide backbone is aligned parallel with the fiber axis.Composed of three or four residues, turns are compact, U-shaped secondary structures stabilized by a hydrogen bond between their end residues. They are located on the surface of a protein, forming a sharp bend that redirects the polypeptide backbone back toward the interior. Glycine and proline are commonly present in turns. The lack of a large side chain in the case of glycine and the presence of a built-in bend in the case of proline allow the polypeptide backbone to fold into a tight U-shaped structure. Without turns, a protein would be large, extended, and loosely packed. A polypeptide backbone also may contain long bends, or loops. In contrast to turns, which exhibit a few defined structures, loops can be formed in many different ways.
(a) The coiled-coil motif (left) is characterized by two or more helices wound around one another. In some DNA-binding proteins, like c-Jun, a two-stranded coiled coil is responsible for dimerization (right). Each helix in a coiled coil has a repeated heptad sequence.
.
The coiled-coil motif comprises two, three, or four amphipathic α helices wrapped around one another. In this motif, hydrophobic side chains project like “knobs” from one helix and interdigitate into the gaps, or “holes,” between the hydrophobic side chains of the other helix along the contact surface. The subunits in some multimeric proteins and in rodlike fibers are held together by coiled-coil interactions. The Ca2+-binding helix-loop-helix motif is marked by the presence of certain hydrophilic residues at invariant positions in the loop. Oxygen atoms in the invariant residues bind a calcium ion through hydrogen bonds. In another common motif, the zinc finger, three secondary structures—an α helix and two β strands with an antiparallel orientation—form a fingerlike bundle held together by a zinc ion. This motif is most commonly found in proteins that bind RNA or DNA.
Additional motifs will be examined in discussions of other proteins. The presence of the same motif in different proteins with similar functions clearly indicates that during evolution these useful combinations of secondary structures have been conserved.
).
A structural domain consists of 100–200 residues in various combinations of α helices, β sheets, turns, and random coils. Often a domain is characterized by some interesting structural feature, for example, an unusual abundance of a particular amino acid (a proline-rich domain, an acidic domain, a glycine-rich domain), sequences common to (conserved in) many proteins (SH3, or Src homology region 3), or a particular secondary-structure motif (zinc-finger motif in kringle domain).
Domains sometimes are defined in functional terms based on observations that the activity of a protein is localized to a small region along its length. For instance, a particular region or regions of a protein may be responsible for its catalytic activity (e.g., a kinase domain) or binding ability (e.g., a DNA-binding domain, membrane-binding domain). Functional domains often are identified experimentally by whittling down a protein to its smallest active fragment with the aid of proteases, enzymes that cleave the polypeptide backbone. Alternatively, the DNA encoding a protein can be subjected to mutagenesis, so that segments of the protein’s backbone are removed or changed (Chapter 7). The activity of the truncated or altered protein product synthesized from the mutated gene is then monitored.
The functional definition of a domain is less rigorous than a structural definition. However, if the three-dimensional structure of a protein has not been determined, identification of functional domains can provide useful information about the protein. Because the activity of a protein usually depends on a proper three-dimensional structure, a functional domain consists of at least one and often several structural domains.
The organization of tertiary structure into domains further illustrates the principle that complex molecules are built from simpler components. Like secondary-structure motifs, tertiary-structure domains are incorporated as modules into different proteins, thereby modifying their functional activities. The modular approach to protein architecture is particularly easy to recognize in large proteins, which tend to be a mosaic of different domains and thus can perform different functions simultaneously.
Epidermal growth factor (EGF) is generated by proteolytic cleavage of a precursor protein containing multiple EGF domains (orange). The EGF domain also occurs in Neu protein and in tissue plasminogen activator (TPA). Other domains, or modules, in these proteins include a chymotryptic domain (purple), an immunoglobulin domain (green), a fibronectin domain (yellow), a membrane-spanning domain (pink), and a kringle domain (blue). [Adapted from I. D. Campbell and P. Bork, 1993, Curr. Opin. Struc. Biol. 3:385.]
). EGF is a small soluble
peptide hormone that binds to cells in the skin and connective tissue, causing them to divide.
It is generated by proteolytic cleavage between repeated EGF domains in the EGF precursor
protein, which is anchored in the cell membrane by a membrane-spanning domain. Six conserved
cysteine residues form three pairs of disulfide bonds that hold EGF in its native conformation.
The EGF domain also occurs in other proteins, including tissue plasminogen activator (TPA), a
protease that is used to dissolve blood clots in heart attack victims; Neu protein, which is
involved in embryonic differentiation; and Notch protein, a cell-adhesion molecule that glues
cells together. Besides the EGF domain, these proteins contain additional domains found in
other proteins. For example, TPA possesses a chymotryptic domain, a common feature in proteins
that catalyze proteolysis.
Note the similarity in the tertiary structures of myoglobin and the two α subunits (blue) and two β subunits (purple) of hemoglobin. The planar white (or gray) structure in the center of each polypeptide chain is the heme prosthetic group. [Myoglobin adapted from S. E. V. Phillips, 1980, J. Mol. Biol.142:531; hemoglobin adapted from B. Shaanan, 1983, J. Mol. Biol.171:31; courtesy of S. Choe.
). Although the sequences of the two proteins were unknown at the time,
Perutz proposed that the similar arrangement of α helices in the two proteins is a
consequence of their having similar amino acid sequences. Later sequencing of myoglobin and
hemoglobin revealed that many identical or chemically similar residues occur in identical
positions throughout the sequences of both proteins. The two proteins also exhibit similar
functions: myoglobin is the oxygen-carrier protein in muscle, and hemoglobin the oxygen-carrier
protein in blood. Most of the conserved residues hold the heme group in place or are
responsible for maintaining the hydrophobic interior of the protein.
As data concerning protein sequences and three- dimensional structures accumulated, the concept that similar sequences fold into similar secondary and tertiary structures was confirmed. The propensity of each amino acid to occur in the various types of secondary structures has been calculated from the amino acid sequence of secondary structures extracted from databases of the three-dimensional structures of proteins. This tabulation of the folding information inherent in the sequence is now being used in attempts to predict the three-dimensional structure of various proteins from their amino acid sequences.
In the classical taxonomy of the eighteenth and nineteenth centuries, organisms were classified according to their morphological similarities and differences. In this century, the molecular revolution in biology has given birth to “molecular” taxonomy: the classification of proteins based on similarities and differences in their amino acid sequences. This new taxonomy provides much information about protein function and evolutionary relationships. If the similarity between proteins from different organisms is significant over their entire sequence, then the proteins are homologs of one another, and they probably carry out similar functions. Sequence similarity also suggests an evolutionary relationship between proteins; that is, they evolved from a common ancestor. We can therefore describe homologous proteins as belonging to the same “family” and can trace their lineage from comparisons of sequences. Closely related proteins have the most similar sequences; distantly related proteins have only faintly similar sequences.
Sequence comparisons have revealed that evolution of the globin proteins parallels the evolution of vertebrates. Major junctions occurred with the divergence of myoglobin from hemoglobin and the later divergence of hemoglobin into the α and β subunits. [Adapted from R. E. Dickerson and I. Geis, 1983, Hemoglobin: Structure, Function, Evolution, and Pathology, Benjamin-Cummings.
). Over time, this ancestral protein slowly
changed, giving rise to myoglobin, which remained a monomeric protein, and to the α
and β subunits, which evolved to associate into the tetrameric hemoglobin molecule. As
the tree diagram in Figure 3-12 shows, evolution of the
globin protein family parallels that of the vertebrates.
The power of such comparative analysis and identification of homologous proteins has expanded substantially in recent years by use of the base sequences in an organism’s genome to deduce the amino acid sequences of the encoded proteins. As discussed in Chapter 7, this approach permits “sequencing” of proteins that are difficult to purify in significant amounts.
A protein is a linear polymer of amino acids linked together by peptide bonds. Various, mostly noncovalent, interactions between amino acids in the linear sequence stabilize a specific folded three-dimensional structure (conformation) for each protein.
).
).Protein tertiary structure results from hydrophobic interactions and disulfide bonds that stabilize folding of the secondary structure into a compact overall arrangement, or conformation. Large proteins often contain distinct domains, independently folded regions of tertiary structure with characteristic structural and/or functional properties.
Quaternary structure encompasses the number and organization of subunits in multimeric proteins.
The sequence of a protein determines its threedimensional structure, which determines its function. In short, function is derived from structure; structure is derived from sequence.
Homologous proteins, which have similar sequences, structures, and functions, most likely evolved from a common ancestor.
