IV.Tertiary Structure of Globular Proteins A.Domains Domains are the fundamental functional and three-dimensional nsof Polypeptide chains thataregr HC-CHa inations of su secondary structural elements(motifs).Folding of the peptide chain CH: within a domain usually occurs independently of folding in othe domnnthe poypeptide chain. CH.Loucine B.Interactions stabilizing tertiary structure Tbeunahreatimensoalstnuctureofeaehpotypeptide amino acid side chains quide the folding of the polvpeptide toform Figure 2.10 1.Disulfide bonds: chains. be separated from each other by many amino acids in the primary sequence of a polypeptide.or may eve 8ntgpowept s into bonding of their side chains.A disulfide bond contributes to the stability of the three-dimensional shape of the protein molecule Glut Aspartate such as immunoglobulins that are secreted by cells. 2.Hydrophobic interactions:Amino acids with nonpolar side chains CH, tend to be located in the interior of the polypeptide molecule 0 tend to be located on the surace of the molecule in contact with the polar solvent.[Note:Recall that proteins located in nonpolar (lipid)environr such as a me mbrane,exhibit the reverse N 3.Hydrogen bonds:Amino acid side chains containing oxygen-or nitrogen-bound hydrogen,such as in the alcohol groups of serine and threo Lysin hydrogen bonds with electron-rich atoms ygen 211:s e al group 16 9r0 Hydrogen bond lonic bond dro bonds between polar groups on the surace of proteins and the aqueous solvent enhances the solubility of the protein. Figure 2.11 mate.can inter ct with n uch as amino group (-NH")in the side chain of lysine(see Figure 2.11)
A. Domains Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are greater 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. B. Interactions stabilizing tertiary structure 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 is a covalent linkage formed from the sulfhydryl group (–SH) of each of two cysteine residues, to produce a cystine residue (Figure 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 polypeptide chains; the folding of the polypeptide chain(s) brings the cysteine residues into proximity, and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the three-dimensional 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. 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 (Figure 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. [Note: Recall that proteins located in nonpolar (lipid) environments, such as a membrane, exhibit the reverse arrangement (see Figure 1.4, p. 4).] In each case, a segregation of R-groups occurs that is energetically most favorable. 3. Hydrogen bonds: Amino acid side chains containing oxygen- or 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 (Figure 2.11; see also Figure 1.6, p. 4). 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 Figure 2.11). IV. Tertiary Structure of Globular Proteins 19 Figure 2.10 Hydrophobic interactions between amino acids with nonpolar side chains. CH2 C CH3 CH3 H C C H N H O Isoleucine Hydrophobic interactions Figure 2.11 Interactions of side chains of amino acids through hydrogen bonds and ionic bonds (salt bridges). CH2 CH2 C O O– CH2 O H CH2 C O O– CH2 CH2 CH2 CH2 +NH3 Glutamate Aspartate Serine Lysine C C H N H O C C H N H O H N C H C O H N C H C O CH2 O H Serine H N C H C O CH2 CH2 CH2 CH2 +NH3 Lysine H N C H C O Hydrogen bond Ionic bond 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 19
20 2.Structure of Proteins C.Protein folding nteracionsbewentesieocheagesntanheaeicemensoal tein Protein folding which occurs within the cell in seconds to minutes,employs a shortcut through the maze otalodngposbrteAiaPepc9nginiranea id side d side attract each other.Conversely.similarly charged side chains repel on the ol not all.possible configurations.seeking a attractions outweigh repulsions.This results in a correctly folded pro- tein with a low-energy state (Figure 2.12). D.Denaturation of proteins aceompanle8yRadoNysndtpeptd%tocr68natihgar。 nts include heat,organic solvents,mechanical mixing.strong acids or hases,ndetergenisnandonsnatheacetnl such a s lead and mer the denaturing agent is removed.However.most proteins.once E.Role of chaperones in protein folding correct pro denatured do not resume their native conformations under favorable er to this prob is that a pro competing folding configurations made available by longer stretches nascent peptide.. me es of proteins proteins-interact with the polypeptide at various stages during the Ielingnpes.nsmechapeesarasmeariantckeseagesor0y ein un SIS IS I as cat proengohensasoihoytogsoatntherovnerPabie.exposed regions do not become tangled in unproductive interactions. V.QUATERNARY STRUCTURE OF PROTEINS and are define polypeptide chains that may be structurally identical or totally unrelated
C. Protein folding Interactions between the side chains of amino acids determine how a long polypeptide chain folds into the intricate three-dimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, employs a shortcut through the maze of all folding possibilities. As a peptide folds, its amino acid side chains are attracted and repulsed according to their chemical properties. For example, positively and negatively charged side chains attract each other. Conversely, similarly charged side chains repel each other. In addition, interactions involving hydrogen bonds, hydrophobic interactions, and disulfide bonds all exert an influence on the folding process. This process of trial and error tests many, but not all, possible configurations, seeking a compromise in which attractions outweigh repulsions. This results in a correctly folded protein with a low-energy state (Figure 2.12). D. Denaturation of proteins Protein denaturation results in the unfolding and disorganization of the protein’s secondary and tertiary structures, which are not accompanied by hydrolysis of peptide bonds. Denaturing agents include heat, organic solvents, mechanical mixing, strong acids or bases, detergents, and ions of heavy metals such as lead and mercury. Denaturation may, under ideal conditions, be reversible, in which case the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins, once denatured, remain permanently disordered. Denatured proteins are often insoluble and, therefore, precipitate from solution. E. Role of chaperones in protein folding It is generally accepted that the information needed for correct protein folding is contained in the primary structure of the polypeptide. Given that premise, it is difficult to explain why most proteins when denatured do not resume their native conformations under favorable environmental conditions. One answer to this problem is that a protein begins to fold in stages during its synthesis, rather than waiting for synthesis of the entire chain to be totally completed. This limits competing folding configurations made available by longer stretches of nascent peptide. In addition, a specialized group of proteins, named “chaperones,” are required for the proper folding of many species of proteins. The chaperones—also known as “heat shock” proteins—interact with the polypeptide at various stages during the folding process. Some chaperones are important in keeping the protein unfolded until its synthesis is finished, or act as catalysts by increasing the rates of the final stages in the folding process. Others protect proteins as they fold so that their vulnerable, exposed regions do not become tangled in unproductive interactions. V. QUATERNARY STRUCTURE OF PROTEINS Many proteins consist of a single polypeptide chain, and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic 20 2. Structure of Proteins Figure 2.12 Formation of secondary structures Formation of domains Formation of final protein monomer Steps in protein folding. 1 2 3 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 20
VI.Protein Misfolding 37 oxygen to one subunit of the tetramer increases the affinity of the other rn subunits for oxygen(see p.29). Extracellula Enzymic cleavage They can arise from different genes or from tis- Enzymic cleavage gnep9echgpro8nsnRoeib9amroancotasne ntracellula VI.PROTEIN MISFOLDING B Protein folding is a complex,trial-and-error process that can sometimes result in improperly folded molecules.These misfolded proteins are usu- proteinscan accumulate.particularly as individ 8ge3geD8posisofhesemisoldedprotansareasocaiedwma number of diseases A.Amyloid disease Spontane mna proiyrc Ghome appndy oo of B-pleated sheets.Accumulation of these insoluble.spontaneously aggregating proteins,called amyloids,has been implicated in many ary n the ade-related amyloid plague that accumulates in Alzheimer disease is amyloid B (AB).a peptide containing 40-42 amino acid residues.X-ray crystal- opy demonstrate ac the ed by pe ag tein expressed on the cell surtace in the prain and other tissues (Figure 2.13).The AB peptides aggregate, amyloid orain par a an cr dis although at least 5-10%of c are familia A econd biologic fac tor invo lved in the development of Alzheimer disease is the accumu- lation ofn Figure 2.13 tangles inside neurons in its healthy version helps in the assembly of the microtubular struc ound ture.The defective t.however,appears to block the actions of its normal counterpart
interactions). Subunits may either 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 p. 29). Isoforms are proteins that 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 isozymes (see p. 65). VI. PROTEIN MISFOLDING Protein folding is a complex, trial-and-error process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see p. 444). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of these misfolded proteins are associated with a number of diseases. A. Amyloid disease 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 conformational state that leads to the formation of long, fibrillar protein assemblies consisting of β-pleated sheets. Accumulation of these insoluble, spontaneously aggregating proteins, called amyloids, has been implicated in many degenerative diseases—particularly in the age-related neurodegenerative disorder, Alzheimer disease. The dominant component of the amyloid plaque that accumulates in Alzheimer disease is amyloid β (Aβ), a peptide containing 40–42 amino acid residues. X-ray crystallography and infrared spectroscopy demonstrate a characteristic β- pleated sheet conformation in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet configuration, 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 Alzheimer disease is derived by proteolytic cleavages from the larger amyloid precursor protein—a single transmembrane protein expressed on the cell surface in the brain and other tissues (Figure 2.13). The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of Alzheimer disease are not genetically based, although at least 5–10% of cases are familial. A second biologic factor involved in the development of Alzheimer disease 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 in its healthy version helps in the assembly of the microtubular structure. The defective τ, however, appears to block the actions of its normal counterpart. Figure 2.13 Formation of amyloid plaques found in Alzheimer disease. Amyloid protein precursor Aβ Intracellular Cell membrane Extracellular Enzymic cleavage Enzymic cleavage Amyloid Cell membrane A B C Photomicrograph of amyloid plaques in a section of temporal cortex from a patient with Alzheimer disease Model of amyloid fibrils Spontaneous aggregation to form insoluble fibrils of β-pleated sheets VI. Protein Misfolding 21 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 21
22 2.Structure of Proteins B.Prion disease TmepnonproenPhasobemgrncg20pinieaethm6ang spongiform encephalopathy in cattle (popularly called"mad cow dis ecies that spn8tcompeeaanheecl6benlcieea8a.PTRsn8e8spd eonierctouseP ble aggreg surface of neuron is a host prote No pri protein.The key to becoming infectious apparently ies in changes in It has been observed bEoaaencaacore2eteessangoagt℃ -sheets in the 2.14.1 is re rbly this m the nor ager al of a n al thus ar verting the normal protein to the pathogenic conformation.The TSEs are invariably fatal,and no treatment is currently available that can alter this outcome VII.CHAPTER SUMMARY Central to understanding protein structure is the concept of the native contemeo n (Figure 2. 15),which is the functio tructu mined by its primary structure.that is.its amino acid sequence Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary,tertiary,and(sometimes) 扬场扬 d to on.a d group of proteins nm of the protein's structure,which are not accompanied by hydrolysis of peptide bonds.Denaturation may be reversible or.more commonly,irre inero inoeoue om versible.Disease can occu r when an apparently normal proteir s in the se me ncluding Cr lako normal pro teins.after abnormal chemical processing.take on a unique conforma Figure 2.14 tional state that leads to the formation of neurotoxic amyloid protein onngo ppoad snootsncemate s an altered version of a normal prion prote ein that for converting normal protein to the pathogenic conformation
B. Prion disease The prion protein (PrP) has been strongly implicated as the causative agent of transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called “mad cow disease”).1 After an extensive series of purification procedures, scientists were astonished to find that the infectivity of the agent causing scrapie in sheep was 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. It has been observed that a number of α-helices present in noninfectious PrPC are replaced by β-sheets in the infectious form (Figure 2.14). It is presumably this conformational difference that 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. VII. CHAPTER SUMMARY Central to understanding protein structure is the concept of the native conformation (Figure 2.15), which is the functional, fully-folded protein structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary structure, that is, its 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. In addition, 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 are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease and the transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease. In Alzheimer disease, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid protein assemblies consisting of β-pleated sheets. In TSEs, 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. 22 2. Structure of Proteins Figure 2.14 One proposed mechanism for multiplication of infectious prion agents. Infectious PrPSc (contains β-sheets) Infectious PrPSc (contains β-sheets) This results in an exponential increase of the infectious form. 3 Infectious PrPSc (contains β-sheets) 1 Interaction of the infectious PrP molecule with a normal PrP causes the normal form to fold into the infectious form. Non-infectious PrPC (contains α-helix) Non-infectious PrPC (contains α-helix) Non-infectious PrPC (contains α-helix) 2 These two molecules dissociate, and convert two additional noninfectious PrP molecules to the infectious form. 1See Chapter 31 in Lippincott’s Illustrated Reviews: Microbiology for a more detailed discussion of prions. INFO LINK 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 22
VIl.Chapter Summary 23 Hierarchy of protein structure composed of Primary can be Fibrous Globular Chaperones B-Sheet B-Bends (reverse turns) d b Non-repetitive structures Supersecondary structures Native ermines e Hydrogen bonds utes t shape Disulfide bonds Denaturants Hydrogen bonds ●Organic solvents Eoctostaticinteractons lead to n the prote leads to Loss of function Altered folding leads to denaturan Figure 2.15 Key concept map for protein structure
VII. Chapter Summary 23 Microbiology Lippincott's Illustrated Reviews α-Helix β-Bends (reverse turns) Non-repetitive structures Supersecondary structures Hydrogen bonds Electrostatic interactions Creutzfeldt-Jakob disease Alzheimer disease Loss of secondary and tertiary structure can be consists of unfolding caused by Fibrous or Globular lead to leads to Loss of function Irreversible denaturation Hierarchy of protein structure Figure 2.15 Key concept map for protein structure. Hydrophobic interactions Hydrogen bonds Electrostatic interactions Disulfide bonds stabilized by Altered folding Amyloid proteins Prions leads to leads to may lead to composed of Chaperones For example: • Catalysis • Protection • Regulation • Signal transduction • Storage • Structure • Transport Biologic function determines leads to lead to lead to leads to β-Sheet Hydrophobic interactions Denaturants Primary is sequence of amino acids Tertiary is the threedimensional shape of the folded chain Quaternary is the arrangement of multiple polypeptide subunits in the protein can form some may regain most proteins cannot refold upon removal of denaturant Native conformation stabilized by may contribute to folding assisted by For example: • Urea • Extremes of pH, temperature • Organic solvents contributes to contributes to Secondary is regular arrangements of amino acids located near to each other in primary structure contributes to 168397_P013-024.qxd7.0:02 Protein structure 5-20-04 2010.4.4 11:31 AM Page 23