Proteins

α-Helix Several different polypeptide helices are found in nature, but the α-helix is the most common. It is a rigid, right-handed spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component L-amino acids extending outward from the central axis to avoid interfering sterically with each other (Fig. 2.6). A very diverse group of proteins contain α-helices. For example, the keratins are a family of closely related, rigid, fibrous proteins whose structure is nearly entirely α-helical. They are a major component of tissues such as hair and skin. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule found in muscles (see Chapter 3 Section II. B.). 1. Hydrogen bonds: An α-helix is stabilized by extensive hydrogen bonding between the peptide bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Fig.  2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the –NH group of a peptide linkage four residues ahead in the polypeptide. This ensures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix. 2. Amino acids per turn: Each turn of an α-helix contains 3.6 amino acids. Thus, amino acids spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix. Figure 2.5 Overlapping of peptides produced by the cleavage action of trypsin and cyanogen bromide. 3. Amino acids that disrupt an α-helix: The R group of an amino acid determines its propensity to be in an α-helix. Proline disrupts an α-helix because its rigid secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Glycine, with hydrogen as its R group, confers high flexibility. Additionally, amino acids with charged or bulky R groups, such as glutamate and tryptophan, respectively, and those with a branch at the β-carbon, the first carbon in the R group (e.g., valine), are less likely to be found in an α-helix. B. β-Sheet The β-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Fig.  2.7A). Because the surfaces of β-sheets appear to be folded or to form “pleats,” they are often called β-pleated sheets. Pleating results from successive α-carbons being slightly above or below the plane of the sheet. Illustrations of protein structure often show β-strands as broad arrows (Fig. 2.7B). Figure 2.6 Structure of an α-helix. 1. Formation: A β-sheet is formed by two or more peptide chains (β-strands) aligned laterally and stabilized by hydrogen bonds between the carboxyl and amino groups of amino acids that either are far apart in a single polypeptide (intrachain bonds) or are in different polypeptide chains (interchain bonds). The adjacent β-strands are arranged either antiparallel to each other (with the N-termini alternating as shown in Fig. 2.7B) or parallel to each other (with the N-termini together as shown in Fig. 2.7C). On each β-strand, the R groups of adjacent amino acids extend in opposite directions, above and below the plane of the β- sheet. β-sheets are not flat and have a right-handed curl (twist) when viewed along the polypeptide backbone. 2. Comparing α-helices and β-sheets: In β-sheets, the βstrands are almost fully extended and the hydrogen bonds between the strands are perpendicular to the polypeptide backbone (see Fig.  2.7A). In contrast, in α-helices, the polypeptide is coiled and the hydrogen bonds are parallel to the backbone (see Fig. 2.6). The orientation of the R groups of the amino acid residues in both the α-helix and the β-sheet can result in formation of polar and nonpolar sides in these secondary structures, thereby making them amphipathic. C. β-Bends β-bends, also called reverse turns and β-turns, reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules and often include charged residues. β-bends were given this name because they often connect successive strands of antiparallel β-sheets. They are generally composed of four amino acids, one of which may be proline, the amino acid that causes a kink in the polypeptide chain. Glycine, the amino acid with the smallest R group, is also frequently found in β-bends. βbends are stabilized by the formation of hydrogen bonds between the first and last residues in the bend. D. Nonrepetitive secondary structure Approximately one-half of an average globular protein is organized into repetitive structures, such as the α-helix and βsheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not random but rather simply have a less regular structure than those described above. The term “random coil” refers to the disordered structure obtained when proteins are denatured (see Chapter 2 Section IV. D.). A Hydrogen bonds perpendicular to the peptide backbone R-C-H ...... H-N H 024 N C / H-C-R R-C-H C N-H H 0 B-strands almost fully extended B N-terminal C-terminal C-terminal N-terminal Antiparallel ẞ-pleated sheet Figure 2.7 A: Structure of a β-sheet. B: An antiparallel β-sheet with the β-strands represented as broad arrows. C: A parallel β-sheet formed from a single polypeptide chain folding back on itself. E. Supersecondary structures (motifs) Globular proteins are constructed by combining secondary structural elements including α-helices, β-sheets, and coils, producing specific geometric patterns, or motifs. These form primarily the core (interior) region of the molecule. They are connected by loop regions (e.g., β-bends) at the surface of the protein. Supersecondary structures are usually produced by the close packing of side chains from adjacent secondary structural elements. For example, α-helices and β-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8. Figure 2.8 Common structural motifs involving α-helices and β-sheets. The names describe their schematic appearance. Motifs may be associated with particular functions. Proteins that bind to DNA contain a limited number of motifs. The helix–loop–helix motif is an example found in a number of proteins that function as transcription factors (see Chapter 31). IV. TERTIARY STRUCTURE The primary structure of a polypeptide chain determines its tertiary structure. The term tertiary refers both to the folding of domains (the basic units of structure and function; see A. below) and to the final arrangement of domains in the polypeptide. The tertiary structure of globular proteins in aqueous solution is compact, with a high density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydrophilic groups are generally found on the surface of the molecule. A. Domains Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are more than 200 amino acids in length generally consist of two or more domains. The core of a domain is built from combinations of supersecondary structural elements (motifs). Folding of the peptide chain within a domain usually occurs independently of folding in other domains. Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain. Figure 2.9 Formation of a disulfide bond by the oxidation of two cysteine residues, producing one cystine residue. O2 = oxygen. B. Stabilizing interactions The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure. The following four types of interactions cooperate in stabilizing the tertiary structures of globular proteins. 1. Disulfide bonds: A disulfide bond (–S–S–) is a covalent linkage formed from the sulfhydryl group (−SH) of each of two cysteine residues to produce a cystine residue (Fig. 2.9). The two cysteines may be separated from each other by many amino acids in the primary sequence of a polypeptide or may even be located on two different polypeptides. The folding of the polypeptide(s) brings the cysteine residues into proximity and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the threedimensional shape of the protein molecule and prevents it from becoming denatured in the extracellular environment. For example, many disulfide bonds are found in proteins such as immunoglobulins that are secreted by cells. It should be noted that protein disulfide isomerase breaks and reforms disulfide bonds during folding. Figure 2.10 Hydrophobic interactions between amino acids with nonpolar side chains. 2. Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids (Fig. 2.10). In contrast, amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with the polar solvent. In each case, a segregation of R groups occurs that is energetically most favorable. 3. Hydrogen bonds: Amino acid side chains containing oxygenor nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine, can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond (Fig. 2.11; see also Fig. 1.6). Formation of hydrogen bonds between polar groups on the surface of proteins and the aqueous solvent enhances the solubility of the protein. 4. Ionic interactions: Negatively charged groups, such as the carboxylate group (−COO− ) in the side chain of aspartate or glutamate, can interact with positively charged groups such as the amino group (−NH3 + ) in the side chain of lysine (see Fig. 2.11). C. Protein folding Interactions between the side chains of amino acids determine how a linear polypeptide chain folds into the intricate threedimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, involves nonrandom, ordered pathways. As a peptide folds, secondary structures form, driven by the hydrophobic effect; hydrophobic groups come together as water is released. These small structures combine to form larger structures. Additional events stabilize secondary structure and initiate formation of tertiary structure. In the last stage, the peptide achieves its fully folded, native (functional) form characterized by a low-energy state (Fig. 2.12). Some biologically active proteins or segments thereof lack a stable tertiary structure and are referred to as intrinsically disordered proteins. Figure 2.11 Interactions of side chains of amino acids through hydrogen bonds and ionic bonds (salt bridges). D. Protein denaturation Denaturation results in the unfolding and disorganization of a protein’s secondary and tertiary structures without the hydrolysis of peptide bonds. Denaturing agents include heat, urea, organic solvents, strong acids or bases, detergents, and ions of heavy metals such as lead. Denaturation may, under ideal conditions, be reversible, such that the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins remain permanently disordered once denatured. Denatured proteins are often insoluble and precipitate from solution. E. Chaperones in protein folding The information needed for correct protein folding is contained in the primary structure of the polypeptide. However, most denatured proteins do not resume their native conformations even under favorable environmental conditions. This is because, for many proteins, folding is a facilitated process that requires ATP hydrolysis and a specialized group of proteins, referred to as molecular chaperones. The chaperones, also known as heatshock proteins (HSPs), interact with a polypeptide at various stages during the folding process. Some chaperones bind hydrophobic regions of an extended polypeptide and are important in keeping the protein unfolded until its synthesis is completed (e.g., Hsp70). Others form cage-like macromolecular structures composed of two stacked rings. The partially folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (e.g., mitochondrial Hsp60). Chaperones, then, facilitate correct protein folding by binding to and stabilizing exposed, aggregation-prone hydrophobic regions in nascent and denatured polypeptides, preventing premature folding. V. QUATERNARY STRUCTURE While many proteins consist of a single polypeptide chain and are defined as monomeric proteins, other proteins consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is the quaternary structure of the protein. Subunits are held together primarily by noncovalent interactions including hydrogen bonds, ionic bonds, and hydrophobic interactions. Subunits may function independently of each other or may work cooperatively, as in hemoglobin, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen (see Chapter 3 Section II. E. 1.). Isoforms of particular proteins all perform the same function but have different primary structures. They can arise from different genes or from tissue-specific processing of the product of a single gene. If the proteins function as enzymes, they are referred to as isoenzymes (see page 70.). 1 Formation of secondary structures 2 Formation of domains Figure 2.12 Steps in protein folding (simplified). VI. PROTEIN MISFOLDING Protein folding is a complex process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see Chapter 19 Section II.). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of misfolded proteins are associated with a number of diseases. A. Amyloid diseases Misfolding of proteins may occur spontaneously or be caused by a mutation in a particular gene, which then produces an altered protein. In addition, some apparently normal proteins can, after abnormal proteolytic cleavage, take on a unique conformation that leads to the spontaneous formation of long, fibrillar protein assemblies consisting of β-pleated sheets. Accumulation of these insoluble fibrous protein aggregates, called amyloids, has been implicated in neurodegenerative disorders such as Alzheimer disease (AD) and Parkinson disease. The dominant component of the amyloid plaque that accumulates in AD is amyloid β (Aβ), an extracellular peptide containing 40 to 42 amino acid residues with a β-pleated sheet secondary structure in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet conformation, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic of the disease. The Aβ that is deposited in the brain in AD is derived by enzymatic cleavages (by secretases) from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues (Fig. 2.13). The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of AD are not genetically based, although at least 5% of cases are familial. A second biologic factor involved in the development of AD is the accumulation of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form of the tau (τ) protein which is hyperphosphorylated and insoluble; in its healthy version, τ helps assemble and stabilize the microtubule structure. The defective τ protein appears to block the actions of its normal counterpart. In Parkinson disease, amyloid is formed from α-synuclein protein. A Amyloid precursor protein Extracellular Ce membrane Intracellular Enzymic cleavage (ẞ-secretase) Enzymic cleavage (y-secretase) B Amyloid Amyloid ẞ (Aß) membran C Spontaneous aggregation of Aß to form amyloid (insoluble fibrils of B-pleated sheets) Figure 2.13 A–C: Formation of amyloid plaques found in Alzheimer disease (AD). (Note: Mutations to presenilin, the catalytic subunit of γ-secretase, are the most common cause of familial AD.) B. Prion diseases Prions or proteinaceous infectious particle are associated with certain diseases. The prion protein (PrP) is the causative agent of transmissible spongiform encephalopathies (TSEs), including Creutzfeldt–Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy, popularly called “mad cow disease,” in cattle. The infectivity of the agent causing scrapie in sheep is associated with a single-protein species that was not complexed with detectable nucleic acid. This infectious protein is designated PrPSc (Sc = scrapie). It is highly resistant to proteolytic degradation and tends to form insoluble aggregates of fibrils, similar to the amyloid found in some other diseases of the brain. A noninfectious form of PrPC (C = cellular), encoded by the same gene as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational modifications have been found between the normal and the infectious forms of the protein. The key to becoming infectious apparently lies in changes in the three-dimensional conformation of PrPC Research has demonstrated that a number of α-helices present in noninfectious PrPC are replaced by β-sheets in the infectious form (Fig. 2.14). This conformational difference is presumably what confers relative resistance to proteolytic degradation of infectious prions and permits them to be distinguished from the normal PrPC in infected tissue. The infective agent is, thus, an altered version of a normal protein, which acts as a template for converting the normal protein to the pathogenic conformation. The TSEs are invariably fatal, and no treatment is currently available that can alter this outcome. 2 1 Interaction of the infectious PrP molecule with a normal PrP causes the normal form to fold into the infectious form. Noninfectious PrPC (contains α-helix) Infectious PrpSc (contains B-sheets) Infectious PrPSc (contains ẞ-sheets) These two molecules dissociate and convert two additional noninfectious PrP molecules to the infectious form. Figure 2.14 One proposed mechanism for multiplication of infectious prions. PrP = prion protein; PrPc = prion protein cellular; PrPSc = prion protein scrapie. VII. Chapter Summary A protein’s native confirmation is the functional, fully folded protein structure (Fig. 2.15). The unique three-dimensional structure of a protein is determined by its primary structure, or amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary, tertiary, and sometimes quaternary structures, which cooperate in stabilizing the native conformation of the protein. A specialized group of proteins named chaperones is required for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein’s structure, which is not accompanied by hydrolysis of peptide bonds. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease (AD) and the transmissible spongiform encephalopathies (TSE), including Creutzfeldt–Jakob disease. In AD, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β-pleated sheets. In TSE, the infective agent is an altered version of a normal prion protein that acts as a template for converting normal protein to the pathogenic conformation.