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ch3 - Amino Acids Peptides and Proteins STEP-BY-STEP GUIDE...

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Unformatted text preview: Amino Acids, Peptides, and Proteins STEP-BY-STEP GUIDE Major Concepts Amino acids share a common structure. There are 20 standard amino acids found in proteins and they are usualiy referred to by either a three-letter or a one-letter code. Each anuno acid contains a car- boxyl group, an amino group, and a hydrogen atom bonded to a central carbon atom, the or carbon. The a carbon is chiral when the R group is anything other than a hydrogen. Therefore, Mth the exception of glycine, there are two enantiomers of each anuno acid, a Inform and an L-forrn. Proteins contain only the L isomers of amino acids. The dififerent chemicat properties of amino acids are the result of the difierent properties of their R groups, which are the basis for categorizing amino acids as '_ nonpotar aromatic, polar; positively charged, or neg~ ati'oeiy charged. Amino acids ionize in aqueous solution. Both the carboxyl and the airline groups can ionize, and amino acids are capable of acting as both weak acids (proton donors) and weak bases (proton accep- __ tors). The R groups of some amino acids can also ion - . ize, affecting overali acid—base behavior. Ail of these ionizable groups affect the net charge on each amino -'-'acid. Consequently, amino acids are often character- _- ized by the pH at which they have no net charge; this f'fcharacteristic is their isoelectric point, or p1. Cit/mere of amino acids are polypeptides or proteins. Amino acids in these poiymers are covalently linked through a peptide bond formed by a condensation re— action between the carboxyl group of one amino acid and the amino group of a second amino acid. in gen— era}, proteins are very large molecules generaliy con- ?Jflmg more than 100 amino acid residues. Peptides neraily contain fewer than 100 amino acid residues. Small peptides perform a number of biological fund tions such as interceilular signaling. Some proteins contain non-amino acid prosthetic groups, which new aily play an important role in the protein’s bioiogical function. Proteins can be studied using a variety of techniques. Proteins can be purified on the basis of solubility, size (sizeexclusion chromatography, gel electrophore- sis), shape or binding characteristics (affinity chro matography), or charge (anion or cation~exchange chromatography, isoelectric focusing). Often, purifica- tion requires multiple steps, each step taking advan— tage of a different characteristic of the protein being purified. A protein’s function depends on its primary structure, which can be determined experimentally. Protein structure is described in terms of four Eevels: primary (1°), secondary (2°), tertiary (3°), and qua- ternary (4"). Primary structure is the specific number and sequence of amino acids in a specific protein. The iocation of disulfide bonds is also a part of the primary structure of a protein. The primary structure deter~ nunes how a protein wili fold into its threewdimensional, functional form. Altering the primary structure in crit~ icai areas can therefore affect the protein‘s function. Primary structure is specified by DNA coding regions; therefore, DNA sequence information provides a fast and accurate method for determining the amino acid sequence of a protein. Alternatively, the amino acid sequence can be determined directiy by first breaking disulfide bonds, cleaving the protein into smali frag— ments, sequencing the fragments by the Edman degradation procedure, and then reconstructing the amino acid sequence of the intact protein, 21 22 Chapter 3 Amino Acids, Peptides, and Proteins Proteins that have simiiar functions in dififierent 0 What are buffers? How do they work? How can a species have similar amino acid sequences. titration curve tell you at what pH a molecule will Typically, stretches of amino acid sequences are invariant in the same proteins from different species. These invariant residues are usually found in func— tionally critical regions. act as an efiective buffer (Fig. 2—17)? Use this in» formation to interpret the titration curve of glycine in this chapter (Fig. 3—10). Using models if necessary, satisfy yourself that enantiorners are not superhnposable and cannot function as identical molecules (Fig. 8—3). 0 Review condensation reactions. The formation of a peptide bond is just one of many examples of mono- mers ioining to form polymers through condensew tion reactions. - Review what a positive free-energy change means in terms of the spontaneity of a reaction (pp. 22m24). Does this mean that an endergonic reaction cannot occur? If not, what circumstances must prevail in or- der for such a reaction to occur? What to Review Answering the following questions and reviewing the relevant concepts, which you have already studied, should make this chapter more understandable. t Review the chemical properties of the biologicaliy important functional groups (Fig. 1ml5), especially —NH§, and ~COO‘. How do these functionat groups behave in aqueous solution (pp. 47—50)? 0 Be sure you understand the difference between a strong acid or base and a weak acid or base (pp. 5669). into which category do ~COO" and leifg groups fit? Topics for Discussion Answering each of the following questions, especially in the context of study group dis- cussions, should help you understand the important points of the chapter. 3.1 Amino Acids Amino Acids Share Gammon Structural Features 1. Why is glycine not optically active? 2. Both the three—letter and one—letter abbreviations for the 20 standard amino acids are very commordy used in the biochemical literature and should be committed to memory. Use the Worksheet in the Self—Test to help you memorize the various amino acid struc- tures, names, and abbreviations. Use “amino acid quiz” as a search term to find interactive quizzes on the Internet. The Amino Acid Residues in Proteins Are L-Stereoisamers 3. Couid a life form that evolved on a planet where all proteins were made up of n—amino acids survive on Earth? Why or why hot? (More on this topic in Chapter 4.) Step~By~Step Guide 23 Amino Acids Can Be Classified by R Group 4. The structures and R-gronp classifications of the 20 standard amino acids are essential parts of a working vocabulary for biochemistry, and should be committed to memory. Look at different amino acid structures and be sure you can identify the or carbon, the carboxyl group, the amino group, and the R group in each. 5. Which amino acid it groups are capable of forming hydrogen bonds? Which will promote hydrophobic interactions? Which amino acid fits into both of these categories? 6. Web anuno acid allows the most structural flexibility when found in a protein? Winch allows the least? Which is the only amino acid that forms disulfide bridges? '3. Does the ability of some amino acids to absorb ultraviolet light translate into a useful technique for the detection of all proteins? Why or why not? 8. What are the pit}. values of R groups on the charged amino acids (see Table 3—K)? Which amino acid has an R group with a pKa value near the pH of most living systems? Box 3-l Absorption of light [7 y Molecules: The lamberr-Beor Law 9. The measurement of protein concentration is a common laboratory procedure. Mea~ surements of light absorption by unknown samples are compared to a standard curve that can be generated by measuring the absorption of solutions with known amounts of protein. Why must these standard solutions be made in the same solvent and at the same pH as that of the unknown protein solutions? Uncommon Acids Also Ha va Important Functions 10. Proteins can contain nonstandard amino acids that are modified versions of the 20 amino acids discussed in this chapter. Do these modifications occur before or after the amino acids are incorporated into the protein molecule? Are amino acids always found incorporated into polypeptide chains in cells? Amino Acids Can Act as Acids and Bases El. How does the zwitterionic nature of individual anuno acids in solution affect their sole- bility in water? Why must individnal amino acids be water soluble? (Note that although R groups vary in water solubility, individually all amino acids having a single amino group and a single carboxyl group are zwitterionic in neutral aqueous solution.) ' 12. Are amino acids weak or strong acids and bases? Amino Acids Ha ve Characteristic Titration Curves 13. Over what pH range (3) is glycine an effective buffer? Over what pH range (s) is glycine not at all an effective buffer? 24 Chapter 3 Amino Acids, Peptides. and Proteins titration Curves Predict the Electric Charge of Amino Acids 14. At appromrnetely What pH would you expect glycine to have a net charge of — 1? Amino Acids Differ in Their Acid-Base Properties 15. Note that the pi values for glutamate and histidine are not the average of the pit; val— ues for each of their three ioniza‘ole groups. Can these pl values be calculated as the av- erage of some set of pifa values? Does this calculation hold for pl values of the other charged amino acids? 3.2 Peptides and Proteins Peptides Are Chains of Amino Acids 16. What is lost from amino acids in the formation of a peptide bond? _4 Go to wwwrschborg and type “i X y2" in the search box. Click on Display Molecule and use Rasmol or Swiss-PDB viewer to view the peptide oxytocin. Click on the MDL logo to dis- play 3. drop-down menu. Try Color or Display to verify that this is an octapeptide. Peptides Can Be Distinguished by Their ionization Behavior 17. Is the tetrapeptide Ale-Glu~Gly-Lys (see Fig. 3—15) 3 good buffer at pH 7.0? Will it move in an electric field at pH 7.0? Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes and Compositions 18. List some functions of small hioactive peptides. 19. Provide an example of a very large protein and a very small protein. In vestigating Proteins with Mass Spectrometry 20. Why is the partial sequencing of protein fragments by mass spectrometry such a useful tool for modern proteome research? 21. How might the number, sequence, and properties of amino acids in a protein affect its structure and function? Some Proteins Contain Chemical Groups Other Than Amino Acids 22. Name five or six different types of prosthetic groups that can be attached to proteins. Step-By-Step Guide 25 23. If you divide the molecuiar weights of the proteins listed in ri‘able 3—2 by the average MW of an amino acid residue, you will find that the number of residues thus caicuiated does not always agree with that reported. What are two possible explanations for this dis— crepancy‘? 3.3 Working with Proteins Proteins Can Be Separated and Purified As you read the rest of this chapter you Wili be able to answer the foilowing questions about protein purification. 24. Which protein purification and separation techniques separate proteins on the basis of size? 25. What technique might you use to separate two proteins that comigrate on an 8138 poly~ acryiamide gel? 26. Which techniques separate proteins on the basis of charge? 27. Which techniques separate proteins on the basis of solubility? 28. Which techniques separate proteins on the basis of binding specificity? ‘ Proteins Can Be Separated and Characterized by Electrophoresis 29. What does a single band on a protein gel represent? 30. What is SDS, and Why is it used in eiectrophoretic procedures? 31. Explain Why two-dimensional gel electrophoresis is a more sensitive analytical method than one-dimensionai gel electrophoresis. Unseparated Proteins Can Be Quantified 32. When you finish this chapter, make a iist of different methods for quantifying the amount of a protein in a complex solution, such as a biood sample or tissue homogenate. 33. When scientists study the activities of proteins such as the enzyme chymotrypsin it is important to be able to measure the amount of a protein. Why? { 26 Chapter 3 Amino Acids, Peptides. and Proteins 3.4 The Structure of Proteins: Primary Structure The Function of 3 Protein Depends on Its Amino Acid Sequence 34. How does information about primary structure contribute to an understanding of a pro— tein’s motion? The Amino Acid Sequences of Millions of Proteins Have Been Determined 35. Use the amino acid sequence of insulin (Fig. 3—24) to quiz yourself: What are the one— 1etter abbreviations and R«group ciassifications for each amino acid listed? Short Polypeptides Are Sequenced with Automated Procedures 36. Outline the individual steps required to sequence a protein. Large Proteins Must Be Sequencer! in Smaller Segments 37. What general steps must be taken before iarger proteins can be sequenced using auto- mated procedures? 38. Placing peptide fragments in their proper order is like piecing together a puzzle. Be sure to understand the example in Figure 3437; there is a similar puzzle in the Self—Test. Amino Acid Sequences Can Also Be Deduced by Other Methods 39; What information concerning the primary structure of a protein would not be availabie from the DNA sequence? Small Peptides and Proteins Can Be Chemically Synthesized 40. Explain how the use of a solid support for chemical synthesis of polypeptides eliminates a step of traditional organic synthesis. Amino Acid Sequences Provide Important Biochemical information 41. Return to an earlier question (#34) and now expand on your answer: in What ways does information about primary structure contribute to an understanding of a protein's function? Protein Sequences Ban Elucidate the History of Life on Earth 42. When using the primary sequences of proteins to study evoiution, one can often obtain nusleading information; Why would comparisons of some proteins not provide useful information? 43. What constitutes a conservative substitution for an amino acid residue in a protein? Step-By-Step Guide 27 Box 3—-3 Consensus Sequences and Sequence Lagos 44. In Figure 1b, What does the presence of the large, red D at position 1 suggest about the characteristics of this end of the protein sequence? What does [iLVFYW} indicate? Discussion Questions for Study Groups Refer to Tobie 3M1. Note the pig values for the u—COOH on the R group of aspartate and glutamate. Why are these pKa values higher than those of ——COOH groups bonded to the or carbon of amino acids? my is the abiiity to synthesize large proteins a useful too: for understanding the structure and function of pro« teins such as enzymes? How might you use this capability to study the function of an enzyme? What are the reasons that proteins with very similar functions might be coded for by very different DNA sequences? The consensus sequence CeXCZ-e)-C-X(3)-F-X(5)—L—X—(2)—H—X(3)-H represents a portion of a DNA binding pro— tein that forms a finger—like projection. This structure is stabilized by the binding of a zinc ion to four of the amino acids in this sequence. Which anuno acids are iikely to be crucial to the stability of this loop and why? Is this 100;) a secondary or tertiary structure? What key term from this chapter describes the zinc ion? Hint: go to wwwrcsborg and iook and up “4211f ” to see What a “zinc finger” structure looks like. Winch of the foilowing consensus sequences might you expect to find in a paraiog of the zinc finger protein just discussed? C—XC4)-C-X(i2)~C«X3«C or C~X4—C-X(2)~H—X3—C 28 Chapter 3 Amino Acids, Peptides. and Proteins SELF-TEST Worksheet The foflowing worksheet helps you memorize the structures, one— and three—letter abbreviations, and R—group cetegory of each 211111110 acid. The first example is filled in as a guide. (Note: many students find it useful to make “flash cards” of the amino acid structures in order to facilitate memorization.) 1 letter 3 letter Group 1 letter 3 letter COO— COO— + l + l HsN’Eli"H H3Nw(i)wH e2 9H2 9H2 e2 r e CH3 0K2 -. m; 1 letter 3 letter 1 letter 3 letter Group 3 letter SeEf—Test 29 30 Chapter 3 Amine Acids, Peptides, and Proteins 000‘ + I mN—?~H ea CH /\ CEg cmg 1 letter 3 letter Group + H3N m" COO C *H SELF-TEST Do You Know the Terms? 1. 4. 11. 12. 13. 15. 16. 1'7. 2. 3. ACROSS The lipid portion of a lipoprotein is known as a group. A covalent bond between two nonadjacent cysteines in a polypeptide chain is a bond. An exempts of a(n) amino acid is histiriine, which can either accept protons or donate them at a pH that is ciose to physiological pH values. All stereoisomers must have at ieast one center. out is a technique that selectively precipitates some proteins, while others remain in solution. Ammonium sulfate ((NH4)2804) is often used for this purpose. The bond type that forms the primary structure. The pH at which the numbers of positive and negative charges on an amino acid are equal is referred to as the point or pl. A single unit Within a polymer: for exampie, lysine in a protein molecule. At pH ’3, any amino acid with an uncharged R group is a _. Proteins from different species that have simi» lar amino acid sequences and functions are re- ferred to as m- C 3 news A reagent used in electrophoresis to separate poiypep» tides on the basis of mass. (abbr.) Many types of separation can be done using this chromatographic technique; its advantage lies in the reduction of transit time on the coiurnn, iimiting diffusional spreading of protein bands and improving resoiution. (soon) After “salting out“ proteins, removal of excess ammonium sulfate can be accomplished by of the protein-salt solution overnight against large volumes of buffer. The whoie assortment of proteins in an organism; f: analogous to the genome. fill-III.- 7. 8. 10. 14. Seif—Test 3 I IIIHIIIIII III-III... >4 p—n ()3 N H as "III-II H n ill-III.- A linear chain of amino acid residues that usually has a moiecular weight less than 10,000 daltons. The degradation procedure provides information about a protein’s primary structure. hisulin obtained from sheep can be used to treat human diabetics because sheep and human insuiin are proteins. Hemogiobin, which contains two sets of identical subunits, is often referred to as a(n) 32 Chapter 3 Amino Acids, Peptides, and Proteins 00 You Know the Facts? 1. Draw the chemical structures of the following amino acids. Give their one- an R»group classification. (a) Alanine d three—letter abbreviations, and (b) Proline (c) Cysteine (d) Phenylalanine (e) Lysine (f) Aspartate 2. Name the following structures. Give their one- and three-letter abbreviations, and R«group classifications. (a) (300” . (b) 000 Hafi”%“3 nafiwéwn (EH2 HM“ (ism-on (If 1‘ng 033 fimN (6) C00" ((1) COO“ HSIGMEEJWH H3fi”(%mfl (€32 (III—12 (£232 (EH2 c112 coo— NH 2: 2 as 1st 3. Name the amino acidCs) described. (3) Provides the least amount of steric hindrance in proteins. (b) Positively charged at physiological pH. (c) Aromatic R group; hydrophobic and neutral at any pH. (d) Saturated hydrocarbon R group; important in hydrophobic interactions. . 10. Self-Test 33 (e) The oniy amino acid having an R group with a pit;I near 7; important in the active site of some enzymes. (f) The only amino acid with a substituted (ii-amino group; influences protein folding by forcing a bend in the chain. ' What is the amino acid sequence of the peptide abbreviated KQNY? . What is the amino acid sequence of the peptide abbreviated RLWEQ? What is the onedetter abbreviation for the amino acid that has an aromatic R group capable of forming hydrogen bonds? What is the onedetter abbreviation for the amino acid that has a sulfur-containing R group and is able to form a disuliide bridge? Which of the following statements describe(s) the peptide bond? (More than one answer may be correct.) A. It is the only covalent bond between amino acids in polypeptides. B. It is a substituted amide linkage. C. It is formed in a condensation reaction. B. It is formed in an exergonic reaction. E. It is unstable under physioiogical conditions. Which of the fotlowing explains Why all individual amino acids are soluble in water but not all peptides are soluble? A. individual amino acids are zwitterions at physiological pHs. B. Ali peptides are insoluble in ...
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