Molecules: Structures and Dynamics

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CHAPTER 3 Molecules: Structures and Dynamics

This chapter describes the properties of water, proteins, nucleic acids, and carbohydrates as they pertain to cell biology. Chapter 7 covers lipids in the context of biological membranes.

Water

Water is so familiar that its role in cell biology and its fascinating properties tend to be neglected. Water is the most abundant and important molecule in cells and tissues. Humans are about two thirds water. Water is not only the solvent for virtually all cellular compounds but also a reactant or product in thousands of biochemical reactions catalyzed by enzymes, including the synthesis and degradation of proteins and nucleic acids and the synthesis and hydrolysis of adenosine triphosphate (ATP), to name a few examples. Water is also an important determinant of biological structure, as lipid bilayers, folded proteins, and macromolecular assemblies are all stabilized by the hydrophobic effect derived from the exclusion of water from nonpolar surfaces (see Fig. 4-5). Additionally, water forms hydrogen bonds with polar groups of many cellular constituents ranging in size from small metabolites to large proteins. It also associates with small inorganic ions.

Physical chemists are still trying to understand water, one of the most complex liquids. The molecule is roughly tetrahedral in shape (Fig. 3-1A), with two hydrogen bond donors and two hydrogen bond acceptors. The electronegative oxygen withdraws the electrons from the O—H covalent bonds, leaving a partial positive charge on the hydrogens and a partial negative charge on the oxygen. Hydrogen bonds between water molecules are partly electrostatic because of the charge separation (induced dipole) but also have some covalent character, owing to overlap of the electron orbitals. The strength of hydrogen bonds depends on their orientation, being strongest along the lines of tetrahedral orbitals. One can think of oxygens of two water molecules sharing a hydrogen-bonded hydrogen. Given two hydrogen bond donors and acceptors, water can be fully hydrogen-bonded, as it is in ice (Fig. 3-1C). Crystalline water in ice has a well-defined structure with a complete set of tetragonal hydrogen bonds and a remarkable amount (35%) of unoccupied space (Fig. 3-1D).

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Figure 3-1 water. A, Space-filling model and orientation of the tetrahedral electron orbitals that define the directions of the hydrogen bonds. B, Tetrahedral local order in liquid water revealed by a theoretical calculation of a three-dimensional map of regions around the central water molecule where the local density of oxygen is at least 40% higher than average. Two adjacent water oxygens are centered near the two hydrogen bond donors, and two other waters are positioned in an elongated cap so that their protons can hydrogen-bond with the central water oxygen. C, Stick figure of crystallized ice showing the tetrahedral network of hydrogen bonds.D, A space-filling model of crystalline ice showing the large amount of unoccupied space. E, Shell of water molecules around a potassium ion. Small ions, such as Li+, Na+, and F−, bind water more tightly than do larger ions, such as K+, Cl−, and I−.

(D–E, From www.nyu.edu/pages/mathmol/library/water, Project MathMol Scientific Visualization Lab, New York University. See “ice.pdb” and “waterbox.pdb.”)

Neither theoretical calculations nor physical observations of liquid water have revealed a consistent picture of its organization. When ice melts, the volume decreases by only about 10%, so liquid water has considerable empty space too. The heat required to melt ice is a small fraction (15%) of the heat required to convert ice to a gas, in which all the hydrogen bonds are lost. Because the heat of melting reflects the number of bonds broken, liquid water must retain most of the hydrogen bonds that stabilize ice. These hydrogen bonds create a continuous, three-dimensional network of water molecules connected at their tetrahedral vertices, allowing water to remain a liquid at a higher temperature than is the case for a similar molecule, ammonia. On the other hand, because liquid water does not have a well-defined, long-range structure, it must be very heterogeneous and dynamic, with rapidly fluctuating regions of local order and disorder. This incomplete picture of water structure limits our ability to understand macromolecular interactions in an aqueous environment.

The properties of water have profound effects on all other molecules in the cell. For example, ions organize shells of water around themselves that compete effectively with other ions with which they might interact electrostatically (Fig. 3-1E). This shell of water travels with the ions, governing the size of pores that they can penetrate. Similarly, hydrogen bonding with water strongly competes with the hydrogen bonding that occurs between solutes, including macromolecules. By contrast, water does not interact as favorably with nonpolar molecules as it does with itself, so the solubility of nonpolar molecules in water is low, and they tend to aggregate to reduce their surface area in contact with water. Such nonpolar interactions are energeti-cally favorable because they reduce unfavorable interactions of nonpolar groups with water and increase favorable interactions of water molecules with each other. This is called the hydrophobic effect (see Fig. 4-5). These interactions of water dominate the behavior of solute molecules in an aqueous environment, where they influence the assembly of proteins, lipids, and nucleic acids into the structures that they assume in the cell. On the other hand, strategically placed water molecules can bridge two macromolecules in functional assemblies.

Proteins

Proteins are major components of all cellular systems. This section presents some basic concepts about protein structure that help to explain how proteins function in cells. More extensive coverage of this topic is available in biochemistry books and specialized books on protein chemistry.

Proteins consist of one or more linear polymers called polypeptides, which consist of various combinations of 20 different amino acids (Figs. 3-2 and 3-3) linked together by peptide bonds (Fig. 3-4). When linked in polypeptides, amino acids are referred to as residues. The sequence of amino acids in each type of polypeptide is unique. It is specified by the gene encoding the protein and is read out precisely during protein synthesis (see Fig. 18-8). The polypeptides of proteins with more than one chain are usually synthesized separately. However, in some cases, a single chain is divided into pieces by cleavage after synthesis.

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Figure 3-2 the 20 l-amino acids specified by the genetic code. Shown for each are the full name, the three-letter abbreviation, the single-letter abbreviation, a stick figure of the atoms, and a space-filling model of the atoms in which hydrogen is white, carbon is black, oxygen is red, nitrogen is blue, and sulfur is yellow. For all, the amino group is protonated and carries a +1 charge, whereas the carboxyl group is ionized and carries a −1 charge. The amino acids are grouped according to the side chains attached to the α-carbon. These side chains fall into three subgroups. Top, The aliphatic (G, A, V, L, I, C, M, P) and aromatic (Y, F, W) side chains partition into nonpolar environments, as they interact poorly with water. Middle, The uncharged side chains with polar hydrogen bond donors or acceptors (S, T, N, Q, Y) can hydrogen-bond with water. Bottom, At neutral pH, the basic amino acids K and R are fully protonated and carry a charge of +1, the acidic amino acids (D, E) are fully ionized and carry a charge of −1, and histidine (pK: ∼6.0) carries a partial positive charge. All the charged residues interact favorably with water, although the aliphatic chains of R and K also give them significant nonpolar character.

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Figure 3-4 the polypeptide backbone. This perspective drawing shows four planar peptide bonds, the four participating α-carbons (labeled 1 to 4), the R groups represented by the β-carbons, amide protons, carbonyl oxygens, and the two rotatable backbone bonds (φ and ø). The dotted lines outline one amino acid.

(Adapted from Creighton TE: Proteins: Structure and Molecular Principles. New York, WH Freeman and Co, 1983.)

Polypeptides range widely in length. Small peptide hormones, such as oxytocin, consist of as few as nine residues, while the giant structural protein titin (see Fig. 39-7) has more than 25,000 residues. Most cellular proteins fall in the range of 100 to 1000 residues. Without stabilization by disulfide bonds or bound metal ions, about 40 residues are required for a polypeptide to adopt a stable three-dimensional structure in water.

The sequence of amino acids in a polypeptide can be determined chemically by removing one amino acid at a time from the amino terminus and identifying the product. This procedure, called Edman degradation, can be repeated about 50 times before declining yields limit progress. Longer polypeptides can be divided into fragments of fewer than 50 amino acids by chemical or enzymatic cleavage, after which they are purified and sequenced separately. Even easier, one can sequence the gene or a complementary DNA (cDNA) copy of the messenger RNA for the protein (Fig. 3-16) and use the genetic code to infer the amino acid sequence. This approach misses posttranslational modifications (Fig. 3-3). Analysis of protein fragments by mass spectrometry can be used to sequence even tiny quantities of proteins.

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Figure 3-16 The sequence of a purified fragment of DNA is rapidly determined by in vitro synthesis (see Fig. 42-1) using the four deoxynucleoside triphosphates plus a small fraction of one dideoxynucleoside triphosphate. The random incorporation of the dideoxy residue terminates a few of the growing DNA molecules every time that base appears in the sequence. The reaction is run separately with each dideoxynucleotide, and fragments are separated according to size by gel electrophoresis (see Fig. 6-5), with the shortest fragments at the bottom. A radioactive label makes the fragments visible when exposed to an X-ray film. The sequence is read from the bottom as indicated. An automated method uses four different fluorescent dideoxynucleotides to mark the end of the fragments and electronic detectors to read the sequence.

(Based on original data from W-L. Lee, Salk Institute for Biological Studies, San Diego, California.)

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Figure 3-3 modified amino acids. Protein kinases add a phosphate group to serine, threonine, tyrosine, histidine, and aspartic acid (not shown). Other enzymes add one or more methyl groups to lysine, arginine, or histidine (not shown); a hydroxyl group to proline; or an acetate to the N-terminus of many proteins. The reducing environment of the cytoplasm minimizes the formation of disulfide bonds, but under oxidizing conditions within the membrane compartments of the secretory pathway (see Chapter 21), intramolecular or intermolecular disulfide (S—S) bonds form between adjacent cysteine residues.

Properties of Amino Acids

Every student of cell biology should know the chemical structures of the amino acids used in proteins (Fig. 3-2). Without these structures in mind, reading the literature and this book is like spelling without knowledge of the alphabet. In addition to their full names, amino acids are frequently designated by three-letter or single-letter abbreviations.

All but one of the 20 amino acids commonly used in proteins consist of an amino group, bonded to the α-carbon, bonded to a carboxyl group. Proline is a variation on this theme with a cyclic side chain bonded back to the nitrogen to form an imino group. Both the amino group (pK > 9) and carboxyl group (pK = ∼4) are partially ionized under physiological conditions. With the exception of glycine, all amino acids have a β-carbon and a proton bonded to the α-carbon. (Glycine has a second proton instead.) This makes the α-carbon an asymmetrical center with two possible configurations. The l-isomers are used almost exclusively in living systems. Compared with natural proteins, proteins constructed artificially from d-amino acids have mirror-image structures and properties.

Each amino acid has a distinctive side chain, or R group, that determines its chemical and physical properties. Amino acids are conveniently grouped in small families according to their R groups. Side chains are distinguished by the presence of ionized groups, polar groups capable of forming hydrogen bonds and their apolar surface areas. Glycine and proline are special cases, owing to their unique effects on the polymer backbone (see later section).

Enzymes modify many amino acids after their incorporation into polypeptides. These posttranslational modifications have both structural and regulatory functions (Fig. 3-3). These modifications are referred to many times in this book, especially reversible phosphorylation of amino acid side chains, the most common regulatory reaction in biochemistry (see Fig. 25-1). Methylated and acetylated lysines are important for chromatin regulation in the nucleus (see Fig. 13-3). Whole proteins such as ubiquitin or SUMO can be attached through isopeptide bonds to lysine e-amino groups to act as signals for degradation (see Fig. 23-8) or endocytosis (see Fig. 22-16).

This repertoire of amino acids is sufficient to construct millions of different proteins, each with different capacities for interacting with other cellular constituents. This is possible because each protein has a unique three-dimensional structure (Fig. 3-5), each displaying the relatively modest variety of functional groups in a different way on its surface.

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Figure 3-5 a gallery of molecules. Space-filling models of proteins compared with a lipid bilayer, transfer rna, and dna, all on the same scale.

(Modified from Goodsell D, Olsen AJ: Soluble proteins: Size, shape, and function. Trends Biochem Sci 18:65–68, 1993.)

Architecture of Proteins

Our knowledge of protein structure is based largely on X-ray diffraction studies of protein crystals or nuclear magnetic resonance (NMR) spectroscopy studies of small proteins in solution. These methods provide pictures showing the arrangement of the atoms in space. X-ray diffraction requires three-dimensional crystals of the protein and yields a three-dimensional contour map showing the density of electrons in the molecule (Fig. 3-6). In favorable cases, all the atoms except hydrogens are clearly resolved, along with water molecules occupying fixed positions in and around the protein. NMR requires concentrated solutions of protein and reveals distances between particular protons. Given enough distance constraints, it is possible to calculate the unique protein fold that is consistent with these spacings. In a few cases, electron microscopy of two-dimensional crystals has revealed atomic structures (see Figs. 7-8B and 34-5).

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Figure 3-6 protein structure determination by x-ray crystallography. A small part of an electron density map at 1.5-Å resolution of the cytoplasmic T1 domain of the shaker potassium channel from Aplysia. The chicken-wire map shows the electron density. The stick figure shows the superimposed atomic model.

(Based on original data from M. Nanao and S. Choe, Salk Institute for Biological Studies, San Diego, California.)

Each amino acid residue contributes three atoms to the polypeptide backbone: the nitrogen from the amino group, the α-carbon, and the carbonyl carbon from the carboxyl group. The peptide bond linking the amino acids together is formed by dehydration synthesis (see Fig. 17-10), a common chemical reaction in biological systems. Water is removed in the form of a hydroxyl from the carboxyl group of one amino acid and a proton from the amino group of the next amino acid in the polymer. Ribosomes catalyze this reaction in cells. Chemical synthesis can achieve the same result in the laboratory. The peptide bond nitrogen has an (amide) proton, and the carbon has a double-bonded (carbonyl) oxygen. The amide proton is an excellent hydrogen bond donor, whereas the carbonyl oxygen is an excellent hydrogen bond acceptor.

The end of a polypeptide with the free amino group is called the amino terminus or N-terminus. The numbering of the residues in the polymer starts with the N-terminal amino acid, as the biosynthesis of the polymer begins there on ribosomes. The other end of a polypeptide has a free carboxyl group and is called the carboxyl terminus or C-terminus.

The peptide bond has some characteristics of a double bond, owing to resonance of the electrons, and is relatively rigid and planar. The bonds on either side of the α-carbon can rotate through 360 degrees, although a relatively narrow range of bond angles is highly favored. Steric hindrance between the β-carbon (on all the amino acids but glycine) and the α-carbon of the adjacent residue favors a trans configuration in which the side chains alternate from one side of the polymer to the other (Fig. 3-4). Folded proteins generally use a limited range of rotational angles to avoid steric collisions of atoms along the backbone. Glycine without a β-carbon is free to assume a wider range of configurations and is useful for making tight turns in folded proteins.

Folding of Polypeptides

The three-dimensional structure of a protein is determined solely by the sequence of amino acids in the polypeptide chain. This was established by reversibly unfolding and refolding proteins in a test tube. Many, but not all, proteins that are unfolded by harsh treatments (high concentrations of urea or extremes of pH) will refold to regain full activity when returned to physiological conditions. Although many proteins are flexible enough to undergo conformational changes (see later discussion), polypeptides rarely fold into more than one final stable structure. Exceptions with medical importance are prions and amyloid (Box 3-1).

BOX 3-1 Protein Misfolding in Amyloid Diseases

Misfolding of diverse proteins and peptides results in spontaneous assembly of insoluble amyloid fibrils. Such pathological misfolding is associated with Alzheimer’s disease, transmissible spongiform encephalopathies (such as “mad cow disease”), and polyglutamine expansion diseases (such as Huntington’s disease, in which genetic mutations encode abnormal stretches of the amino acid glutamine). Accumulation of amyloid fibrils in these diseases is associated with slow degeneration of the brain. Pathological misfolding also results in amyloid deposition in other organs such as the endocrine pancreas in Type II diabetes. The precursor of a given amyloid fiber may be the wild-type protein or a protein modified through mutation, proteolytic cleavage, posttranslational modification, or polyglutamine expansion. The pathology of amyloidosis is not well understood. Some, but not all, amyloids are intrinsically toxic to cells. Some amyloid precursors are more toxic than the fibrils themselves. In all cases, fibril initiation is very slow, but once formed, fibrils act as seeds to promote the assembly of additional protein into fibrils.

Given that many unrelated proteins and peptides form amyloid, it is remarkable that most of these twisted fibrils have similar structures: narrow sheets up to 10μm long consisting of thousands of short β-strands that run across the width of the fibril. The β-strands can be either parallel or antiparallel, depending on the particular protein or peptide. Some amyloid fibrils consist of multiple layers of β-strands. The structures of the various parent proteins have nothing in common with each other or with amyloid cross β-sheets, so these are rare examples of polypeptides with two stable folds. To form amyloid, the native protein must either be partially unfolded or cleaved into a fragment with a tendency to aggregate.

In the common form of dementia called Alzheimer’s disease, the peptide that forms amyloid is a proteolytic fragment of a transmembrane protein of unknown function called β-amyloid precursor protein. “Infectious proteins” called prions cause transmissible spongiform encephalopathies. Normally, these proteins do no harm, but once misfolded, the protein can act as a seed to induce other copies of the protein to form insoluble amyloid-like assemblies that are toxic to nerve cells. Such misfolding rarely occurs under normal circumstances, but the misfolded seeds can be acquired by ingesting infected tissues.

Other proteins, including the peptide hormone insulin, the actin-binding protein gelsolin, and the blood-clotting protein fibrinogen, form amyloid in certain diseases. An inherited point mutation makes the secreted form of gelsolin susceptible to cleavage by a peptide processing protease in the trans-Golgi network. Fragments of 53 or 71 residues form extracellular amyloid fibrils in several organs.

Given that amyloid fibrils form spontaneously and are exceptionally stable, it is not surprising that functional amyloids exist in organisms ranging from bacteria to humans. For example, formation of the pigment granules responsible for skin color depends on a proteolytic fragment of a lysosomal membrane protein that forms amyloid fibrils as a scaffold from melanin pigments. Budding yeast has a number of proteins that can either assume their “native” fold or assemble into amyloid fibrils. The native fold of the protein Sup35p serves as a translation termination factor that stops protein synthesis at the stop codon (see Fig. 17-8). Rarely, Sup35p misfolds and assembles into an amyloid fibril. These fibrils sequester all the Sup35p in fibrils, where it is inactive. The faulty translation termination that occurs in its absence has diverse consequences that are inherited like prions from one generation of yeast to the next.

Although proteins fold spontaneously into a unique structure, it is not yet possible to predict three-dimensional structures of proteins from their amino acid sequences unless one already knows the structure of an ortholog or paralog. Then one can use the known structure and the amino acid sequence of the unknown to build a homology model that is often accurate enough to make reliable inferences about function. Predicting protein structures from sequence alone would have profound practical consequences, since the number of protein sequences known from genome-sequencing projects far exceeds the number of established protein structures (about 10,000).

The following factors influence protein folding:

1. Hydrophobic side chains pack very tightly in the core of proteins to minimize their exposure to water. Little free space exists inside proteins, so the hydrophobic core resembles a hydrocarbon crystal more than an oil droplet (Fig. 3-7). Accordingly, the most conserved residues in families of proteins are found in the interior. Nevertheless, the internal packing is malleable enough to tolerate mutations that change the size of buried side chains, as the neighboring chains can rearrange without changing the overall shape of the protein. Interior charged or polar residues frequently form hydrogen bonds or salt bridges to neutralize their charge.
2. Most charged and polar side chains are exposed on the surface, where they interact favorably with water. Although many hydrophobic residues are inside, roughly half the residues that are exposed to solvent on the outer surface are also hydrophobic. Amino acid residues on the surface typically appear to play a minor role in protein folding. Experimentally, one can substitute many residues on the surface of a protein with any other residue without changing the stability or three-dimensional structure.
3. The polar amide protons and carbonyl oxygens of the polypeptide backbone maximize their potential to form hydrogen bonds with other backbone atoms, side chain atoms, or water. In the hydrophobic core of proteins, this is achieved by hydrogen bonds with other backbone atoms in two major types of secondary structures: α-helices and β-sheets (Fig. 3-8).
4. Elements of secondary structure usually extend completely across compact domains. Consequently, most loops connecting α-helices and β-strands are on the surface of proteins, not in the interior (Fig. 3-9). Exceptions are found in some integral membrane proteins (see Figs. 10-3, 10-13, 10-14, and 10-15), where α-helices can reverse in the interior of the protein.
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Figure 3-7 Space-filling (A) and ribbon (B) models of a cross section of the bacterial chemotaxis protein CheY illustrate some of the factors that contribute to protein folding. α-Helices pack on both sides of the central, parallel β-sheet. Most of the polar and charged residues are on the surface. The tightly packed interior of largely apolar residues excludes water. The buried backbone amides and carbonyls are fully hydrogen-bonded to other backbone atoms in both the α-helices and β-sheet. (PDB file: 2CHF.)

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