Amino Acids, Polypeptides, and Proteins

Amino Acids, Polypeptides, and Proteins
Chapter 29 Amino Acids, Polypeptides, and Proteins The spider has three sets of spinnerets that produce protein-containing fluids that harden as they are drawn out to form silk threads. Introduction to General, Organic, and Biochemistry, 10e John Wiley & Sons, Inc Morris Hein, Scott Pattison, and Susan Arena 1

Course Outline 29.1 The Structure-Function Connection
29.2 The Nature of Amino Acids 29.3 Essential Amino Acids 29.4 D-Amino Acids and L-Amino Acids 29.5 Amphoterism 29.6 Formation of Polypeptides 29.7 Protein Structure 29.8 Protein Functions 2

Course Outline 29.9 Some Examples of Proteins and their Structure
Loss of Protein Structure Test for Proteins and Amino Acids Determination of the Primary Structure of Polypeptides Chapter 29 Summary 3

The Structure-Function Connection
Proteins are polymeric substances composed of many amino acids linked together in a unique sequence. Proteins are a major class of food needed for growth and maintenance. Proteins have many different functions including serving as structural materials and as enzymes. 4

The Structure-Function Connection
Proteins are classified as simple or as conjugated. Simple proteins yield only amino acids when hydrolyzed. Conjugated proteins yield amino acids plus at least on additional product. The function of a protein is determined by the unique sequence of amino acid units in the protein and its structural organization. 5

The Nature of Amino Acids
An amino acid is any molecule with at least one carboxyl (COOH) group and one amino group (–NH2). The amino acids found in proteins are called alpha (α) amino acids because the amino group is attached to the first or α-carbon atom adjacent to the carboxyl group. 6

The general formula of α-amino acids is shown below. The variable R group, the carboxyl group and the amino group are attached to the α-carbon atom. 7

The variable R group commonly referred to as the side chain can contain alkyl groups, aromatic groups, –NH2 , –COOH , –OH, and S-containing groups. Amino acids are divided into four groups based on the characteristics of the amino acid side chains as seen on the following slide. This classification has been chosen to emphasize the importance of the side chains in amino acid and protein structure. 8

1. Nonpolar amino acids: The side chains are either aliphatic or aromatic and are hydrophobic. 2. Polar, uncharged amino acids: The side chains contain functional groups with polar bonds such as alcohols or amides and are hydrophilic. 3. Acidic amino acids: The side chain contains a carboxyl group. Also called “negatively charged” amino acids due to their form a physiological pH. 4. Basic amino acids: The side chain contains a nitrogen that can act as a base. Also called “positively charged” amino acids due to their form a physiological pH. 9

Amino acids are classified as basic, neutral, or acidic, depending on whether the ratio of –NH2 to –COOH groups in the molecules is greater than 1:1, equal to 1:1, or less than 1:1, respectively. This ratio differs from 1:1 only if the amino acid side chain (R–) contains an additional amino or carboxyl group. 10

Table 29.1 on the following slides show the classifications, names, abbreviations, and structures of the common amino acids derived from proteins . . . 11

Your Turn! Write the structure of an α-amino acid that has a methyl group as a side chain. 15

Your Turn! Write the structure of an α-amino acid that has a methyl group as a side chain. 16

Your Turn! What is the classification of the amino acid shown below (nonpolar, polar uncharged, acidic or basic)? 17

Your Turn! What is the classification of the amino acid shown below (nonpolar, polar uncharged, acidic or basic)? This amino acid is polar uncharged because it contains a polar hydroxyl group. 18

Your Turn! Which of the following is not attached to the α-carbon of an amino acid that is used to make protein molecules? An α-hydrogen A group containing a phosphorus atom An R-group A carboxylate group An amino group 19

Your Turn! Which of the following is not attached to the α-carbon of an amino acid that is used to make protein molecules? An α-hydrogen A group containing a phosphorus atom An R-group A carboxylate group An amino group Amino acids that are in proteins contain all the groups listed except for a group containing a phosphorus atom. 20

Essential Amino Acids Essential amino acids are those that are required to maintain good health. These amino acids cannot be synthesized in the body and must be supplied from dietary protein. 21

Essential Amino Acids There are ten essential amino acids for humans as shown below. 22

Essential Amino Acids Dietary protein is either complete or incomplete based on the presence or absence of these ten essential amino acids. Complete protein supplies all the essential amino acids. Incomplete protein is deficient in one or more essential amino acids. 23

Your Turn! Which statement best describes essential amino acids?
The twenty amino acids found in proteins. The vitamins that are needed to maintain good health. The amino acids that cannot be synthesized by the organism involved and must be supplied in the diet. The amino acids that are lacking in a vegetarian diet. The amino acids that are just needed by humans. 24

Your Turn! Which statement best describes essential amino acids?
The twenty amino acids found in proteins. The vitamins that are needed to maintain good health. The amino acids that cannot be synthesized by the organism involved and must be supplied in the diet. The amino acids that are lacking in a vegetarian diet. The amino acids that are just needed by humans. The statement in red above best describes amino acids. 25

D-Amino Acids and L-Amino Acids
The alpha carbon of all amino acids (except glycine) is chiral. The two stereoisomers of alanine are shown below. Fischer projection formulas illustrate the D and L configurations of amino acids in the same way they illustrate these configurations in carbohydrates . . . 26

The –COOH group is written at the top of the projection formula. The D configuration is indicated by writing the α-NH2 to the right of carbon 2. The L configuration is indicated by writing the alpha α-NH2 to the left of carbon 2. 27

Although some D-amino acids occur in nature, only L-amino acids occur in proteins. The (+) and (-) signs in the names indicate the direction of rotation of plane-polarized light by the amino acid. 28

Your Turn! How many chiral carbon atoms are in the amino acid shown below? 29

Your Turn! The amino acid has one chiral carbon atom which is shown in red. 30

Amphoterism Amino acids are amphoteric which means they can react as acids or bases. For example alanine can react with strong acid and with strong base. 31

Amphoterism Amino acids exist in neutral solutions as zwitterions.
A zwitterion is a dipolar ion formed by an internal transfer of a proton from a –COOH group to a –NH2 group as seen with alanine below. 32

Amphoterism The zwitterion can react as an acid or base as shown below. 33

Amphoterism The pH at which there is no migration toward an electrode is called the isoelectric point. This occurs since there is no net charge (equal number of positive and negative charges). The isoelectric points of some amino acids are shown below. 34

Amphoterism Isoelectric points are found at pH values ranging from:
7.8 to 10.8 for basic amino acids 4.8 to 6.3 for neutral amino acids 2.8 to 3.3 for acidic amino acids 35

Your Turn! Draw the structure of L-phenylalanine in its zwitterion form and a reaction of this zwitterion with a strong base. 36

Your Turn! Draw the structure of L-phenylalanine in its zwitterion form and a reaction of this zwitterion with a strong base. 37

Formation of Polypeptides
A peptide bond or peptide linkage is an amide bond that is formed when a –COOH group of one amino acid reacts with the α-amino group (–NH2) of a different amino acid. 38

The product formed from two glycine molecules is called glycylglycine (abbreviated Gly–Gly). It is called a dipeptide because it contains two amino acid units. 39

A molecule with three amino acid units is a tripeptide; with four is a tetrapeptide; with five a pentapeptide; and so on. Peptides containing up to about 40–50 amino acid units in a chain are called polypeptides. The units making up the peptide are amino acids, minus the elements of water, and are referred to as amino acid residues. Longer chains of amino acids are proteins. 40

A linear peptide will have one end with a free –NH2 group and another end with a free –COOH group. The end of the polypeptide chain with the –NH2 group is called the N-terminal residue and the end with the free –COOH group is called the C-terminal residue. 41

The sequence of amino acids in a chain is numbered starting with the N-terminal residue, which is written on the left. The C-terminal residue is written on the right. Any segment of the sequence that is not specifically known is placed in parentheses. 42

Peptides are named as acyl derivatives of the C-terminal amino acid. Ala–Tyr–Gly is called alanyltyrosylglycine. 43

Alanine and glycine can form two different dipeptides, Gly–Ala and Ala–Gly. If three different amino acids react six tripeptides can be formed with each amino acids appearing once. 44

There are a number of small, naturally occurring polypeptides with significant biochemical functions. (Over 30 different peptides are known at present.) In general, these substances serve as hormones or nerve transmitters. The amino acid sequence and chain length give a polypeptide its biological effectiveness and specificity. Table 29.4 on the next slide shows some examples of small polypeptides and their functions . . . 45

Your Turn! Write the structure of the tripeptide Ala–Cys–Ser. 47

Your Turn! Write the structure of the tripeptide Ala–Cys–Ser.
First write the structures of the amino acids. 48

Your Turn! Write the structure of the tripeptide Ala–Cys–Ser.
Then link the amino acids with peptide bonds. 49

Your Turn! You are given the two amino acid molecules shown below. How many different dipeptides can you make from these two molecules? Draw these molecules. 50

Your Turn! You are given the two amino acid molecules shown below. How many different dipeptides can you make from these two molecules? Draw these molecules. There are two dipeptides you can form from these two amino acids. 51

Protein Structure In general, for a protein molecule to serve a specific biological function, it must have a defined shape. Chemists typically describe large proteins on four levels. Primary structure Secondary structure 3) Tertiary structure 4) Quaternary structure 52

Protein Structure Primary structure is the amino acid sequence of a protein. The primary structure of a protein affects other levels of protein structural organization. 53

Protein Structure The primary structure of insulin is shown below. Notice that this protein consists of two chains linked by disulfide bonds. 54

Protein Structure The secondary structure of proteins can be characterized as a regular three-dimensional structure held together by hydrogen bonding between the oxygen of the C=O and the hydrogen of the H–N groups in the polypeptide chains. The a-helical and b-pleated-sheet structures are two examples of secondary structure which are shown on the following slides . . . 55

Protein Structure -Helix 56

Protein Structure β-Pleated sheet 57

Protein Structure The tertiary structure of a protein is the shape or conformation of a protein molecule. The overall three-dimensional conformation of the protein molecule is held together by a variety of interactions between amino acid side chains including: Hydrogen bonding Ionic bonding Disulfide bonding 58

Protein Structure The diagram below represents the three principal types of bonding that determine the tertiary structure of a protein molecule. 59

Protein Structure A fourth type of structure, called a quaternary structure, is found in some complex proteins. These proteins are made of two or more smaller protein subunits or polypeptide chains. The quaternary structure refers to the shape of the entire complex molecule and is determined by the way in which the subunits are held together by noncovalent bonds such as hydrogen bonding and ionic bonding among others. 60

Your Turn! Show the hydrogen bonding that can occur between the side chains of the two amino acid residues shown below. 61

Your Turn! Hydrogen bonding can occur between the side chains as shown below. 62

Your Turn! All proteins have primary structure. (True or False) True
63

Your Turn! All proteins have primary structure. (True or False) True
Primary structure refers to the sequence of amino acids. All proteins have amino acids so the statement is True. 64

Protein Functions Proteins are the machinery that makes life possible. Their varied structures allow for a great variation in function. The functions of proteins can be classified in seven different ways . . . 65

Protein Functions 1. Structural support: Some proteins provide structural support like the class of proteins found in connective tissue and skin. 2. Storage: Storage proteins serve as containers for a variety of chemicals needed for life such as the storage protein in the liver that stores iron. 3. Transport: Transport proteins move important chemical between cells. The protein hemoglobin transports oxygen. 66

Protein Functions 4. Defense: Defensive proteins kill or repel other organisms such as immunoglobulins (antibodies) which protect us from bacterial invaders. 5. Motion/movement: Animals are absolutely dependent on muscles for survival. Muscle protein is one example of a protein with a movement function. 6. Regulation: Regulatory proteins have the responsibility for coordinating life’s many processes. Insulin, for example, is a protein hormone that controls blood-glucose levels. 67

Protein Functions 7. Catalysis: Catalytic proteins (enzymes) increases the rate of chemical reactions. Catalytic proteins are needed because metabolic reactions are much too slow to sustain life without the aid of a catalyst. 68

Some Examples of Proteins and their Structures
Fibrous proteins are proteins that have a fiber-like shape. Fibrous proteins serve as structural supports and tend to be water insoluble. α-Keratin found in hair, fibroin found in silk, and collagen found in many tissues are examples of fibrous proteins . . . 69

α-Keratin in an α-helix. 70

Fibroin in an β-pleated sheet. 71

Collagen forms a triple helix. 72

The globular proteins have a characteristic compact, roughly spherical shape. These proteins are capable of very complex functions and have very complex structures. It is the globular proteins that provide most of life’s machinery. Some globular proteins are described on the following slides . . . 73

Myoglobin: This conjugated protein functions as a storage protein, binding oxygen for muscle tissue. Within the center of this structure is an organically-bound iron atom in the heme ring that allows myoglobin to store oxygen. 74

Hemoglobin: This protein is a transport protein found in red blood cells that transport oxygen. This protein has quaternary structure. It is composed of four subunits and an oxygen-binding heme group in each subunit. 75

Carboxypeptidase A: This protein is a catalytic protein (an enzyme) that catalyzes protein digestion in the small intestine. Fatty Acid Binding Protein: This protein is a transport protein which transports fatty acids through the blood stream. Ferritin: This protein is a storage protein in the liver that stores iron. 76

HIV Reverse Transcriptase: This protein is an enzyme. It is responsible for converting the AIDS virus genetic material into a form that can infect the host. This enzyme is a primary target for drugs designed to block the spread of AIDS. Myosin: This protein is a movement protein. It is an important protein in muscle tissue. 77

Human Growth Hormone: This protein is a regulatory protein responsible for coordinating overall growth. Immunoglobulin G: This protein is a defense protein. Immunoglobulins (antibodies) are large binding proteins that serve a critical biological function. These proteins bind molecules that are foreign to the body as a defense against disease. 78

Loss of Protein Structure
The loss of protein structure will affect the protein’s function. If a protein loses only its natural three-dimensional conformation the loss of structure is referred to as denaturation. Denaturation involves the alteration or disruption of the secondary, tertiary, or quaternary-but not primary—structure of proteins. 79

Denaturation may involve reversible alterations caused by a slight shift in pH to the extreme alterations involved in tanning a skin to form leather. Structural change occurs when a protein molecule is denatured. During denaturation the hydrogen, electrostatic, and disulfide bonds are broken, resulting in a change of structure and properties. 80

The figure below shows what happens to a protein molecule during protein denaturation. 81

Loss of protein structure also occurs with hydrolysis of the peptide bonds to produce free amino acids. This chemical reaction destroys the protein’s primary structure. Proteins can be hydrolyzed by boiling in a solution containing a strong acid such as HCl or a strong base such as NaOH. 82

Tests for Proteins and Amino Acids
A number of tests exist for characterizing amino acids, peptides, and proteins which include the five tests shown on the next slide . . . 83

Test Reagent Positive Test Result
Xanthoproteic Test HNO3 Yellow products for proteins with benzene rings Biuret Test CuSO4 Violet color for peptides and proteins with two or more peptide bonds. The test is negative for amino acids and dipeptides. Lowry Assay CuSO4 and molybdates/ tungstates A dark violet-blue for tyrosine or tryptophan-containing proteins. Bradford Assay Coomassie Brilliant Blue dye Dark blue color for proteins. The most sensitive common test for proteins. Ninhydrin Test Ninhydrin A blue solution for all amino acids except proline and hydoxyproline. 84

Complex mixtures of amino acids are readily separated by thin-layer, paper (shown below) or column chromatography. A chromatogram showing separation of several amino acids with paper chromatography is shown below. After chromatography Before chromatography 85

Electrophoresis is used to separate proteins on the basis of a difference in size and a difference in charge. Results of an electrophoresis separation is shown below. 86

Determination of the Primary Structure of Polypeptides
Two methods that can be used to determine the primary structure of a protein are the use of Sanger’s reagent and the Edman degradation . . . 87

Sanger’s reagent, 2-4-dinitrofluorobenzene (DNFB), reacts with the amino group of the N-terminal amino acid of a polypeptide chain. The carbon–nitrogen bond between the amino acid and the benzene ring of Sanger’s reagent is more resistant to hydrolysis than are the remaining peptide linkages. When the substituted polypeptide is hydrolyzed, the terminal amino acid remains with the dinitrobenzene group and can be isolated and identified. 88

The Sanger’s reagent reaction is illustrated in the following equations. 89

The Edman degradation method has been developed to split off amino acids one at a time from the N-terminal end of a polypeptide chain. In this procedure, the reagent, phenylisothiocyanate, is added to the N-terminal amino group. The N-terminal amino acid–phenylisothiocyanate addition product is cleaved from the polypeptide chain which is isolated and identified. The shortened polypeptide chain is then ready to undergo further Edman degradation. 90

The Edman degradation process occurs as follows. 91

Your Turn! What is the amino acid sequence of a octapeptide (eight amino acids) based on the following information. Three rounds of Edman degradation yield sequentially Ser, then Gly, and then His; Hydrolysis yields three fragments: a tripeptide, His–Gly–Thr; another tripeptide, Phe–His–Pro; and a dipeptide, Ser–Gly. 92

Ser–Gly–His–Gly–Thr–Phe–His–Pro
Your Turn! Based on the Edman degradation the first amino acid in the octapeptide is Ser, the second is Gly and the third is His. So the first fragment must be Ser–Gly and the second fragment must be His–Gly–Thr. The last fragment must be Phe–His–Pro. The octapeptide amino acid sequence is: Ser–Gly–His–Gly–Thr–Phe–His–Pro 93

Chapter 29 Summary Proteins are polymers of amino acids. Simple proteins yield only amino acids upon hydrolysis and conjugated proteins yield amino acids and additional products. Amino acids found in proteins are called alpha (α) amino acids. Amino acids are classified as nonpolar, polar uncharged, acidic, or basic. Ten of the common amino acids are essential amino acids because they are needed for good health. 94 94

Chapter 29 Summary The α-carbon is chiral for all the common amino acids except for glycine. Chiral amino acids are L-amino acids or D-amino acids. Only L-amino acids are found in proteins. Amino acids are amphoteric (or amphiprotic) because they can react either as an acid or as a base. The pH at which an amino acid has no overall charge is called the isoelectric point of the amino acid. 95 95

Chapter 29 Summary An amide bond is formed when a carboxyl group on one amino acid reacts with the α-amino group of a second amino acid to eliminate water. Linear peptides have one end with a free amine group (the N-terminal residue) and one end with a free carboxyl group (the C-terminal residue). Commonly, the term protein refers to a peptide that contains 50 or more amino acids. The structure of proteins can be described as primary structure, secondary structure, tertiary structure and quaternary structure. 96 96

Chapter 29 Summary Proteins provide the machinery for life. The functions of proteins are commonly broken down into several different categories: structural, storage, transport, defense, motion/movement, regulation and catalysis. Fibrous proteins have highly developed secondary structures and often function in support roles. Globular proteins have a more complex tertiary structure and a compact shape. 97 97

Chapter 29 Summary Denaturation is the process by which a protein loses its natural three-dimensional conformation. Many colorimetric tests are available for amino acids and proteins such as the xanthoproteic test, buiret test, Lowry assay, Bradford assay, and the ninhydrin test. Two methods to determine the primary structure of a protein are the use of Sanger’s reagent and the Edman degradation. 98 98

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