ch4 - Structure of Proteins STEP-BY-STEP GUIDE Major...

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Unformatted text preview: Structure of Proteins STEP-BY-STEP GUIDE Major Concepts A protein’s conformation, or three—dimmtonal struc- ture, as described by its secondary, tertiary, and qua ternary structure. All of these are dependent on the primary structure. Weak, noncovalent interactions stabiiize these levels of architecture, which determine a protein’s overall shape. Peptide bonds connect amino acid residues in pro— ri‘hese C—mN bonds have some double-bond character, which restricts rotation, limiting the possible confor— mations of a polypeptide chain. The N—Ca and Cow—C bonds can rotate, but their possible conforma- tions are limited by steric hindrance. A Ramachan— dran plot depicts the conformations theoretically permitted for peptides. Secondary structure refers to the arrangement of ad— ;‘Jacent amino acids in regular recurring patterns. Some common secondary structures are the a helix, ,8 conformation, ii turn, and collagen helix. These structures are formed, in part, in response to nonco- _:__va1ent interactions between neighboring amino acids. :. They are stable because hydrogen—bond formation is maximized and steric repulsion is minimized in these conformations. Specific amino acids are more likely to be found in some secondary structures than in others; __ information about the amino acid se- quence of a protein can be used to predict where : Ie’se structures are likely to occur. O o 223 5’) .8 9:? (D it 5.4 structure refers to the actual three by tonal arrangement of a single chain of amino 3:. . linear amino acid sequence is a critical determinant the final shape of a protein. Secondary structure, The Three-Dimensional bend-producing amino acids, and hydrophobic interac- tions also contribute to the folding of a protein molecule and therefore to its overall shape. Protein folding is a compiex process that is stili not well understood. in some cases, protein folding occurs spontaneously, de— termined soleiy by the primary sequence. Weak, non- covalent interactions between individual amino acid residues piay a rote in stabilizing a protein’s tertiary structure. Most soluble proteins are compact, globuiar structures that are somewhat flexible. 'i‘he ability to change conformation is critical to protein function; enzymes, for example, often change shape when they bind substrate. X-ray diffraction techniques have provided direct information on the tertiary structure of many proteins. Quaternaryr structure is a level of protein architece ture found only in proteins having more than one polypeptide chain (subunit). The three—dimensional arrangement of the separate subunits in the protein is referred to as its quaternary structure. Interactions between the individual subunits are stabilized by the same weak, noncovaient interac— tions that help stabilize the secondary and tertiary structure. One welicharacterized protein with quater- nary structure is hemoglobin, which consists of four distinct polypeptide chains. The association of these subunits into an oligorner contributes to the ability of hemoglobin to both bind and release oxygen under the appropriate physiological conditions (see Chapter 5 for more information on hemoglobin function). The qua» ternary structure of fibrous proteins such as coilagen contributes to the tensile strength of these proteins. Very iarge assemblies of polypeptides have also been described; for example ribosomes are supramoiecular assemblies that contain more than 80 proteins. 41 42 Chapter 4 The Three~Dimensional Structure of Proteins What to Review 0 How are peptide bonds formed? Does a polypep— tide have direction (p. 82)? Answering the foilowing questions and reviewing the , Recall that proteins are mafia using Gilly “mum relevant COflCePES’ Wm‘lh you have aheady Stumed’ acids. Draw a representation of an L~ and a Ira—amino should make this chapter more understandable. acid (p. 72). - Protein chemistry takes place in aqueous solution. 0 The amino acid sequence, or primary structure, of Renew your appreciation of the role water plays in a protein influences all other levels of protein ar- all biochemistry discussions (pp. 43—419). chitecture. What bonds and interactions are most 0 Weak, noncovaient interactions are extremely im— importfint in this level 0f DTGEE-‘in Structure? HOW portantinprotein architecture. What are the various can the sequence of amino acids in a polypeptide types of weak interactions common in biomolecules, be datemlifled (Dp- 83, 94-100)? and What are their relative strengths (pp. 50mm)? Topics for Discussion Answering each of the following questions, especialiy in the context of a study group dis- cussion, should help you understand the important points of this chapter. 4.1 Overview of Protein Structure A Protein’s Conformation is Stabilized Largely by Weak Interactions 1. Would the “simple rules” stated at the end of this section (p. 115) be the same it pro- tein chemistry occurred in a nonpolar solvent? The Peptide Bond is Rigid and Planar 2. What makes peptide bonds planar? 3. Why can’t psi (at) and phi (a) both be zero? 4.2 firotein Secondaty Structure The at Helix is a Common Protein Secondary Structure 4. Find the 1p and (is angles for an or helix on the Ramachandran plot shown in Figure 4~3. Are these angles theoreticaliy allowed? 5. Are the side chains of amino acids in an or helix on the outside or inside of the helix? Box 4"? Knowing the Right Hand from the left 6. Attempt to draw for yourself an a helix containing a mixture of s— and e-amino acids. What does this attempt tell you about or helices? Amino Acid Sequence Affects Stability of the or Helix 7. The properties of the various amino acid side chains dictate their interactions in sec- ondary structure elements. in a helices, the properties of the side chains place con- straints on the stabiiity of the helix. ;4'.- '3' Step-By-Step Guide 43 _ Explore an or helix. Go to wwwrcsborg and type “i ALI” into the search box. Click on Display Molecule and seiect either Rasmol Viewer or Swiss-FDR Viewer to display the structure. Click on the MDL logo (or right click on a PC) to View a menu of display opw tions. Choose Display a Ball & Stick; drag molecule to move it around in the window. - Rotate the helix so you are looking down its central axis. Where are the R groups found? 0 What is the amino acid sequence of this or heiix? (Hint: Color —> Amino Acid, then Options we Labels) a Hydrogen bonds are formed at regular intervais between backbone amines and car- bonyls; in terms of linear sequence, how many asuno acid residues lie between two residues invoived in a singte hydrogen bond? (Hint: Options —-> Display —> Hydro- gen Bonds) 0 What other forces stabilize the or helix structure? The 6 Conformation Organizes Polypeptide Chains into Sheets 8. Where are the R groups located in a B sheet? . What noncovalent forces stabilize B~sheet structures? 10. In silk fibroiu, which amino acid residues have functional groups participating in inter- chain l-l bonds? Which have groups extending above and below the plane of the sheet? 11. What are the limitations on the kinds of amino acids found in 6 structures? Compare these to the constraints discussed for the or helix [3 Turns Are Common in Proteins 12. What is unusual about peptide bonds involving the imino nitrogen of proline in ,8 turns? 13. How zrrany amino acid residues are typically found in a 3 turn and how is this structure stabilized? ' Common Secondary Structures Have Characteristic Dinedral Angles 14. Note Where each of the secondary structures falls on the Ramachandran plot in Figure 4-43. Common Secondary Structures Can Be Assessed by Circular Dichroism 15. How might a manager of a biological supply company use circular dichroism to ensure the quality of protein—based products? 3- Protein Tertiary and Quaternary Structures 16. What distinguishes tertiary from quaternary structure in proteins? Do all proteins have quaternary structure? ll -: 44 Chapter 4 The Three-Dimensional Structure of Proteins 17. How couid you determine if a protein has quaternary structure? Fibrous Proteins Are Adapted for 3 Structural Function 18. What contributes to the insoiubility of fibrous proteins such as lac-keratin and now does this insolubility contribute to the function of these proteins? 19. What covalent bonds contribute strength in each of these fibrous proteins: nil—keratin, collagen, eiastin? 20. Wool and silk are both composed of fibrous proteins; wool can stretch and shrink but silk cannot. What is the moiecular basis for the different characteristics of these two fibers? Box 4—2 Permanent Waving ls Biochemical Engineering 21. Why can a badly done perm be so damaging to hair? Box 4—3 Why Sailors, Explorers, and College Students Should Eat Their Fresh Fruits and Vegetables 22. What is the relationship between vitamin C and scurvy? 23. How does vitamin C participate in collagen formation? $3M 4—4 The Protein Data Bank 24. Go to wwrcsborg. and look up 1v4f or icdg to see a collagen triple helix. Click on Display Molecule and use Rasmoi or Swiss-FDR viewers to display the structure. Ciick on the MDL logo and try out different display and coloring options. Structural Diversity Reflects Functional Diversity in Globular Proteins 25. Try to envision how protein structure contributes to the functions of the various glob- ular proteins mentioned in this section. Myoglobin Provided Early Clues about the Complexity of Globular Protein Structure 26. Why does it make functional sense that a globular protein, such as myoglobin, is solu- ble in water? 27. What weak interactions contribute to the close packing and stability of the structure of myoglobin? Step-By-Step Guide 45 Globular Proteins Have a Variety of Tertiary Structures 28. Take a look at Figure 4—21 to see a sampling of the 3—D structures that can be formed from the three basic secondary structures. 29. What is the difference between a domain and a motif? 30. Provide some examples of common protein motifs. Box 4~5 Methods for Determining the Three-Dimensional Structure of a Protein 31. Why can’t you see proteins under a highwpower light microscope? 32. Why must X rays be used to generate “images” of proteins? 33. Why is it important to know about protein structure in aqueous solutions? Protein Motifs Are the Basis for Protein Structural Classification 34. Why are the proteins belonging to a structural “family” likely to be related with respect to their evolution? Protein Quaternary Structures Range from Simple Dimers to Large complexes 35. After reading this section, list some suprarnolecular complexes consisting only of pro— teins and some consisting of proteins associated with other types of biomolecules. 36. What kinds of bonds or interactions would be important in the assembly of large protein complexes? 'Protein Denaturation and Folding ' loss of Protein Structure Results in toss of Function 37. Under what conditions are proteins denatured? 38. What bonds or interactions are disrupted during denaturation? Amino Acid Sequence Determines Tertiary Structure 39. When denatured proteins refoid in the presence of low levels of detergent or denatu- rant, they form many incorrect disulfide bonds. What does this tell you about the process of protein folding? 46 Chapter 4 The Three-Dimensional Structure of Proteins Polypeptides Fold Rapidly by a Stepwise Process 40. What are the two models of protein folding presented in this section? 41. What are the noncovalent interactions that drive folding in each of these models? Some Proteins Undergo Assisted Folding 42. Yeast mutants that are deficient in heat shock proteins {some of which are of molecu— iar chaperones) have proteins that do not fold correctly. One role of molecular Chapar- ones is to bind to nascent protein chains (proteins that are being synthesized) and gore vent associations between newly synthesized regions. Why would this be necessary? Defects in Protein Folding May Be the Molecular Basis for a Wide Range of Human Genetic Disorders 43. Winch type of noncovalent interaction is likely to be responsible for stabilizing amyloid fibrils? 44. Describe two or three cellutar consequences of protein misfolding. Box 46 Death by Misfoiding: The Prion Diseases 45. The conversion of the normal PrP protein to the PrPEc form is the resuit of an atteration that changes the structure of the prion protein. Based on information from this chap- ter, what other changes in a protein‘s amino acid composition would be expected to change a protein’s structure and therefore its function? l‘ ‘ 5 ' I . g . 1' Discussion Questions For Study Groups - Why is the or helix often referred to as a condensed secondary structure, Whereas the ,8 conformation is often called extended? How might this rotate to a protein’s function? 0 Which annno acids will usually be found in the interior of a globular protein and which wiil be found on the ex— terior? Why? Under what circumstances might you find exceptions to this “rule”? t Explore some of the protein structures in this chapter (see iist below). Go to wwwrcsborg and type in the pro— tein name or PDB identification number in the search “endow. Click on Display Molecule and use the Rasmoi or Swiss—PUB viewer to see the structure. Drag the molecule to rotate it; click on the MDL logo to access addi~ tional display commands. Protein PDB ID Aipha—hemolysin 7 AHL Deoxy hemogiob‘m 2 HEB Collagen 1 CGD Globin 1 VRF GroEL/GroES complex 1 AON Myoglobin 1 M80 / 2 MBW Poliovirus 2 PLV Pymvate Kinase 1 PKN ri‘obacco Mosaic Virus 1 WM Troponin 4 TNC SELF-TEST Do You Know the Terms? ACRBSS 1. Celiular agents that assist in protein folding at elevated temperatures. 6. Covalently iinlced amino acids with a singie amino terminus and a single carbonyl temdnus is called aCn) 8. Bonds that occur between cysteine residues in proteins. 9. disc called a “motif.” 11. Hemoglobin is aCn) protein because it has two or more polypeptide chains. 14. They protrude in opposite directions from the zigzag structure of m O H H III-III- Self—Test 47 >—‘ ()7 sh the B conformation. [2 words) S—S—‘i 15. GCKKGGLVCAH for exampie; structure. 17. Muscle fibers are an exampie of a{n) complex. 18. Protein secondary - structure that extends 0.35 run per amino acid . residue. _ 20. Though unrelated based ' on their amino acid sequences, proteins that belong to a(n} related structurai features. . The noncovalent interactions that are thought to be the driving force behind the formation of a "molten globule." An exampie is the reformation of disulfide bonds during permanent waving. a. (3 pa H H ‘4 U1 M have _ - A native protein is in its functional . _An exampie of protein rnisfolding that has lethal - ' consequences. ___ 'I A stable arrangement of a few secondary structures. a heiices are stabilized by bonds between the I "carbonyl oxygen and the amino hydrogen. (3 turn is an example of structure. 1.: Disrupting the hydrophobic interactions of a single Subunit protein wouid have the greatest effect on the 3- structure of that protein. ........J M a U1 10. 12. 13. 16. 19. 21. 22. 24. 25. H O\ . An example of a supramoiecular assembly is the col§a~ gen . nil-keratin is referred to as a supramolemdar complex of protein subunits; hemoglobin with only four subunits is referred to as am) The saddle conformation is 3.00 structure. Myoglobin is to tertiary as hemoglobin is to Roasting a chicken results in the permanent of myosin and actin proteins in the muscle cells. Individual amino acids when polymerized in a protein. The «.6 subunits in hemoglobin compose a single ; the intact hemoglobin tetramer contains two of these. Protein secondary structure that extends 0.15 nm per amino acid residue. This class of proteins binds to and shields hydrophobic portions of unfolded polypeptides in cells. These pro— teins also are denatured by elevated temperatures. Refers to the portion of a protein that is often composed of noncontiguous amino acid sequences and is usually de- fined on the basis of its contribution to protein function. 48 Chapter 4 The Three-nimensional Structure of Proteins Do You Know the Facts? 1. Which of the foliowing statements is/are true con- cerning peptide bonds? A. They are the only covalent bond formed be— tween amino acids in polypeptide structures. 13. The angles between the participating C and N atoms are described by the values psi (4/) and phi (a). C. They have partial double-bond character. D. A and C. E. All of the above are true. In an or helix, the R groups on the amino acid residues: A. are found on the outside of the helix spiral. B. participate in the hydrogen bonds that stabiiize the helix. 0. aliow only right~handed helices to form. D. A and B are true. E. A, B, and G are true. Which of the foilowing bonds or interactions is/are possible contributors to the stabih'ty of the tertiary structure of a globuiar protein? (Hint: remember the amino acid categories.) A. peptide bonds between a metal ion cofactor and a histidine residue B. hydrophobic interactions between histidine and tryptophan R groups C. covalent disulfide crosselinlts between two me— thionine residues D. hydrogen bonds between serine residues and the aqueous surroundings E. Ali of the above contribute. Refer to the following numbered statements to answer questions 4—7. {1) Found in the same percentage in all proteins. (2} Stabilized by H bonds between w—NH and ~00 groups. (3) Found in globular proteins. (4) Affected by amino acid sequence. (5) An extended conformation of the polypeptide chain. (6) Includes all 20 standard ermine acids in equai frequencies. (7') Hydrophobic interactions are responsible for the primary structure. 4. Which statements are true of or heiices? 5. Which statements are true of ,8 sheets? 6. Which statements are true of both or heiices and 6 sheets? 7. 8. 10. 11. 12. 13. Which statements are true of neither or helices nor 6 sheets? Describe and compare the positions of the R groups of amino acids that are in an or helix and in a 6 con- formation. . Fibrous proteins, such as or~l<eratin, coliagen, and eiastin, have evolved to be strong and/or elastic. They are also insoluble in water. What wouid you guess this insolubility means about the spatiai pow sitioning oi the various groups of amino acids that constitute these proteins? What does denaturation mean? Under what condi- tions are proteins denatured? From the types of bonds and interactions below, identify which is most responsibie for the struc- tures described in (a)—-(j}. ionic interactions hydrophobic interactions van der Wants interactions (a) the association of hemoglobin subunits in its quaternary structure (is) primary structure of proteins (c) secondary structure of proteins ((1) the interaction between hemoglobin subunits upon binding oxygen (9) the binding of iron within the heme group (f) an a helix (g) a 6 sheet (h) association of the heme group Within the myoglobin polypeptide (i) the hardness of rhinoceros horn (j) the compactness of the interior of myoglobin covalent bonds hydrogen bonds Refer to Table 3—«2 in the textbook for questions 12 and 13. How many proteins in the table exhibit quaternary structure? How can you tell? A. All B. None C. Half D. Five E. One cannot teli from the data provided. How many proteins in the table exhibit a helix sec— ondary structure? How can you tell? A. All 13. None C. Half D. Two E. One cannot teli from the data provided. Self-Test 49 Applying What You Know 1. Answer the foilowing questions concerning the poiypeptides whose amino acid sequences are shown below. it may help to refer to Table 3—1 for the threedletter abbreviations and to Figure 3W5 for amino acid structures. (1) Glymiiew'i‘rpwlieumllsmite—Phe—Gly—Val—Vai——Ala——Gly—Val~lle—Gly—Trp—lle—Leu—Leu—Ile (2) Gly—Pro—Hyp—Gly—Pro—Met—GIy—Pr0“SermGlyml?ro~Arg~Gly—~i’ro«HypmGly—PromHypmGly (3) Gly~Met~'i‘rp-Pro-Gluwi\/iethys_Gly—Glu—Pro—Ala—His—Val—Arg—Asp—Tyr—Pro—Leu—Leu (4) G1—Met—'i‘rp~Pro—Glu—Met—Cys—Gly—G1unPromnlamHismVal—mArgwAsp—lyr—Oys—Leu—«Leu (3.) Which would be most likely to form an iii—helical structure? (b) Which couid have at least one disulfide bond? ((2) Which would be most likely to be part of a fibrous protein found in cartilage? ((2) Suppose that the first amino acid residue in peptide (i) were altered from Gly to Pro. Would you expect this change to have an effect on the secondary structure of this peptide? Why or why not? 2. Myoglobin and the individuai subunits of hemoglobin are similar, though certainly not identical, in size, overall shape, and function. Would you expect a molecule of myoglobin or a subunit of hemoglobin to have a greater ra- tio of nonpolar to polar amino acids? Why? 3. How would the following agents and/or procedures interfere with or disrupt the different levels of protein archi- tecture? Why? (a) Addition of SDS {see p. 92). (b) Oxidation of cysteine with performic acid to cleave disulfide bonds (Fig. 8—26). (c) Addition of proteases such as trypsin (p. 99). Biochemistry on the Internet .; Chapters 3 and 4 have introduced you to the structural hierarchy of celluiar proteins. The extent to which the linear ' sequence of amino acids, the primary structure, determines the final three-dimensional shape, or tertiary structure, ' of proteins is quite remarkabie. As our understanding of the mechanisms underlying protein folding has expanded, it has become possible to iden- tify unknown proteins and predict their shape, and therefore their function, based only on amino acid sequence in— formation. This type of protein analysis reiies on information that is available from sequence databases around the world. The irey to unlocking this information is some knowledge of the DNA or amino acid sequence of the “mystery” protein. _ ' The problem outiined below provides a key {an amino acid sequence) and directs you to some of the doors (data also access sites) to the ever-expanding amount of scientific information available on the Internet. The list of data- ses presented here is by no means exhaustive; this information is intended only to provide a limited and somewhat structured exposure to protein databases and moiecular modeling. - As you proceed through this exercise you will be able to obtain additionai information by foliowing any of the links not appear during your search. It will be up to you to sift through all this information to find what is relevant to your research, so don’t stray too far in your initial forays and be sure to find your way back. _'Problem the course of evaluating a 6-year-old patient with bronchitis, a systematic hematologicai study of the chiid‘s blood performed. An abnormal protein was detected and additional tests on the patient’s blood sample were performed. EhQS-eitests, which included isoelectric focusing, electrophoresis, and cationwexchange high performance liquid chro- m ,Ogl'aphy, confirmed the existence of a novel blood protein. The protein was also observed in blood samples from _ hild’s father and other blood relatives, indicating a genetic basis for the abnormality. rE‘he abnormal protein was late'd a pure form and the following amino acid sequence was determined: VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSF?ETT KTYFPHFDLS HGSAQVKGHG KKVADALTNA VAHVDDMPNA LSALSDLHAH KLRVDPVNFK LLSHCLLVTL AAHLPAEFTP AVHASLDKFL ASVSTVLTSK YR do is to try to identify this novel blood protein. There are a number of excelient sites for anaiyzing amino acid Ci' rotein sequences on the Internet. Many of these sites use BLAST (Basic Local Aligmnent Search rfool), which ._.a'§e__t__of search engines designed to query available databases. Choose one of the foliowing sites for your analysis. 50 Chapter 4 "fhe Three-Dimensional Structure of i’mteins 0 NCBI Blast search at www.ncbi.nlm.riih.govfblast 0 PIR—lnternationai Protein Sequence Database at For either of these sites, foliow the internal links to locate a sequence comparison engine. For example, at the NCBI site use the “Protein BLAST” links. Type the entire sequence above into the ap« propriate search field and submit your sequence for analysis. Be sure to limit your search to Homo saptens when possible. What does this analysis tell you about the identity of your protein? (1)) The molecular mass of the abnormai biooci protein is approximately 100 daltons more than the mass of the “normai” protein. What does this suggest about the abnormal protein, and what might you do to verify your hypothesis? (c) What information can you find on the secondary, tertiary, and quaternary structure of the “normal” version of this protein? You can start your struc- tural analysis at a variety of Web sites that provide information on the three-dimensional structure of proteins. For this initial analysis try using the RCSB Protein Data Bank at wwwrcsborgzpdb. Perform a keyword search using the protein class name that you identified in part (a). Answers 51 ANSWERS Do You Know the Terms? ACROSS DflWN I. chaperones 1. conformation 6. polypeptide 2. prion 8. disulfide 3. motif 9. foid 4. hydrogen 11. multisubumt 5. secondary 14. Rgroups 7. tertiary 15. primary 9. fibril 17. supramolecflar 10. oligomer 18. .8 conformation 12. supersecondary 20. superfamfly 13. quaternary 23. hydrophobic 16. denaturatiort 26. renaturation 19. residues 21. protomer 22. ahelix 24. hsp 25. domain 52 Do 1. one 9°:‘3 10. 11. 12. Chapter 4 The Three-Dimensional Structure of Proteins You Know the Facts? G. Disuifide bonds between cysteine residues are aiso covalent bonds in polypeptides. Psi {1,0} and phi (p) describe the Col—MC and the NmCa bonds, not those involved in the peptide bond. . A. H bonds form between m—NH and ——GO groups of the polypeptide backbone, not between. R groups. Both left— and right~handed helices can oc~ cur (though extended leftuhanded helices have not been observed in proteins). D. The hydroxyl group on the serine side chain can form H bonds with surrounding water molecules. Peptide bonds occur only between amino acids, not between an amino acid and a cofactor. Histiw dine has no hydrophobic R group. Oniy cysteines can form disulfide linkages. 2, 3, 4 2, 3, 4, 5 2, 3, a l, 6, 7 In an or helix, the polypeptide backbone is tightly wound along the long axis of the molecule; the R groups extend outward from the rod, like spokes, in a helical array. In a .8 conformation, the back- bone of the polypeptide chain is extended into a zigzag rather than a helical rod; the R groups of ad— jacent amino acids protrude in opposite directions from the zigzag structure, creating an alternating pattern when viewed from the side. . These fibrous proteins are an exception to the rule that hydrophobic R groups of amino acid residues must be buried in the interior of a protein; they have a high concentration of hydrophobic amino acids in the interior and on the surface of the protein. Denaturation is the total ioss or randomization of three~dimensional structure. it can be reversible or irreversible. Extremes of heat and pH, exposure to some organic solvents such as alcohol or acetone, or some other substances such as urea or some detergents, will denature proteins. The covalent peptide bonds of proteins are not broken, but de» naturation disrupts the many weak, noncovalent interactions that are most important in maintaining the native three-diinensionai conformation. (a) Hydrophobic interactions; (1)) covalent bonds; (c) hydrogen bonds; (d) ionic interactions; (e) covaient bonds; (f) hydrogen bonds; (g) hydrogen bonds; (it) covalent bonds; (i) covalent bonds; (j) hydrophobic interactions. D. A protein has quaternary structure only if it has more than one polypeptide chain, or subunit. Five of the proteins listed have at least two subunits, and so have quaternary structure. 13. E. None of the data in the table provides any infor- mation regarding secondary structure. Applying What You Know 1. (a) t; (b) 4; (c) 2; (d) No; peptide (1) could adopt an a structure before the change of the amino- terniinai residue to proiine. Though proiine residues are rarely found in 0: helices, the amino terminus is one location where the proline R group would have relatively little effect. The amino (inuno) group would not interfere with the helical structure. . The hemoglobin subunit would have a higher ratio of nonpolar to polar amino acids. In hemoglobin’s native conformation, each subunit is in contact with the other subunits; hydrophobic interactions between the subunits are important in the stability of the tetramer, and nonpolar amino acids can be on the surface of the subunit. The external surface of the myogiobin molecule would have more poiar anuno acid residues, to increase hydrogen bonding with the aqueous surroundings. . {a} 8138, a detergent, would interfere with hyd drophobic and ionic interactions, and would there— fore interfere in secondary, tertiary, and quater— nary structure; primary structure would not be affected. Cb) For proteins with disulfide bridges, oxidation of cysteine with performic acid to cleave disulflde bonds would alter all levels of protein ar~ chitecture. Disulfide bonds are part of primary structure, and a loss of these stabilizing covalent cross~lin1<s would very likely disrupt a protein’s na~ tive conformation. Proteins without disulfide bridges wouid be unaffected. (c) Proteases cleave peptide bonds, destroying primary structure. All other ievels depend on the anuno acid sequence, and so would be affected as well. Biochemistry on the interest (a) When the unknown sequence is compared to known sequences in protein data bases, search en— gmes detect domains that are conserved between the proteins. Limiting the search to Homo sopiens shows a high degree of similarity between the unknown pro- tein and the alpha chain of human hemoglobin. (b) The higher moiecular mass coupied with the sequence siiniiarity to the a: chain of hemoglobin suggests that the protein is a mutant form of hemoglobin that has more amino acids than the normal form. Because the average weight of a single amino acid in a polypeptide chain is around 310, it is possibie that an extra amino acid residue has been inserted into this abnormal hemoglobin subunit. This can be verified by directiy comparing the amino acid sequence of the abnormal protein and the human hemoglobin armchain sequence. The abnormal l-ib identified in the patient contains 142 residues with an additional glutamate (E) inserted after the proline (P) at position 87. In fact, the amino acid sequence of the ab- normal protein provided in the original question is the complete sequence for an a—chain variant called Hb Cantonsville, named after the town where it was first identified. For the complete ref- erence, see Moo—Penn, WE, Swan, D11, Hine, TX, Baine, RM, Jue, D.L., Benson, J.M., Johnson, Mil, Virshup, 31M, and Zinltham, Wit. lib Cantonsville (Glutamic acid inserted between Pro-37 ((32) or and Thr—38 ((33) a) J. Biol. Chem, 26421454, 1989. (c) There are a number of good sites for the Internet-based analysis of protein structure. One excellent site is the Protein Data Bank. Using “glo- bin” as a keyword produces more than 400 related Answers 53 structures while using “compound: hemoglobin and source: human” produces only about 100 related structures. Several molecule display options allow you to see the protein in 3-D, but many require the use of piugins that can be downloaded from the Web (cg, Java). Help with downloads is available on this same page. Still images of the molecule can also be viewed from this page. in most of the 31) viewers, you can see the protein’s secondary structure by setting the Display option to Ribbons. This Will display the protein's beta conformation and a—helical structures. These models are interactive allowing you to manipulate the movement of the proteins by clicking on the structure and then dragging it with your mouse. In the hemoglobin model you should see four subunits composed entirely of o: helices connected by 8 turns. Note the heme moiecules bound to each of the four subunits; this is where hemoglobin binds oxygen. ...
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