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Proteins The Function of Proteins Amino Acids The Peptide Bond Structure of Proteins Myoglobin, Hemoglobin and Oxygen Overview of Protein Structure and Function Effect of Temperature and pH 1 • Proteins are among the “essential” compounds necessary for the normal functioning of a living system. • The name is derived from the Greek word “Proteios”, meaning first. • All proteins are made from amino acids. R O NH2 C C H OH 2 The function of proteins Enzymes Biological catalysts. Antibodies They fight off infection. Transport Move materials around Ex. hemoglobin for O2. Regulatory As hormones, they control metabolism. Structural coverings and support skin, tendons, hair, nails, bone. Movement muscles, cilia, flagella. 3 Proteins are often gigantic in size – Insulin: Molecular Wt = 5700 – Hemoglobin: Molecular Wt = 64,000 – Virus Proteins: Molecular Wt = 40,000,000 4 Amino acids All these proteins are made from the same building blocks. • • • Twenty common amino acids. All are -amino acids except proline. A primary amine is attached to the carbon. -carbon - the carbon just after the acid. H | R-C-COOH | NH2 5 Amino acids Because an acid and base are both present, an amino acid can form a +/- ion, called a zwitterion. H | R-C-COOH | NH2 H | R-C-COO | NH3+ How well it happens is based on pH and the type of amino acid. 6 -Amino acids Except for glycine, the carbon is attached to four different groups - it is a chiral center. For Carbohydrates We used the D- form. For Amino Acids We use the L- form. COO+ | H3N - C - H | R The amino group is on the left side of the fischer projection. 7 Classification of amino acids • The -amino acid group is the same in each of the amino acids. • They are classified by the polarity of the side chain (R). Hydrophobic - water fearing non-polar side chains Hydrophilic - water loving polar, neutral chains negatively charged positively charged 8 Neutral, nonpolar side chains glycine H | H-C-COO| +NH 3 leucine H 3C H \ | HC-CH2-C-COO/ | +NH H3C 3 H3C valine H \ | HC-C-COO/ | H3C +NH3 alanine H | CH3-C-COO| +NH 3 9 Neutral, nonpolar side chains H 3C H | | H3C-CH2-CH-C-COO| +NH isoleucine 3 H | CH3 -S-CH2-CH2-C-COO| +NH 3 methionine H | -CH2-C-COO| phenylalanine +NH3 proline H 2C | H 2C CH-COO| +NH 2 H 2C 10 Polar, neutral amino acids H serine | HO-CH2-C-COO| +NH 3 tyrosine HO- H | -CH2-C-COO| +NH 3 HO H | | CH3-CH-C-COO| +NH 3 threonine tryptophan N H | CH2-C-COO| +NH 3 11 Polar, neutral amino acids H | HS-CH2-C-COO| +NH 3 cysteine O H || | H2N-C-CH2-CH2-C-COO| +NH 3 glutamine O H || | H2N-C-CH2-C-COO| +NH 3 asparagine 12 Acidic, polar side chains Based on having a pH of 7. glutamic acid O H || | -O-C-CH -CH -C-COO2 2 | +NH 3 aspartic acid O H || | -O-C-CH -C-COO2 | +NH 3 13 Basic, polar side chains Based on a pH of 7. +NH H | | H2N-C-N-CH2-CH2-CH2-C-COO| +NH arginine 3 2 H | + H3N-CH2-CH2-CH2-CH2-C-COO| +NH lysine 3 H | CH2-C-COO| +NH N H 3 histidine H H N + 14 Essential Amino Acids • Proteins are constantly being produced in the body for growth and repair. • Of the 20 amino acids found in these proteins, 10 cannot be synthesized by the body. • Arginine* • Histidine * • Isoleucine • Leucine • Lysine Methionine Phenylalanine Threonine Tryptophan Valine 15 • Histidine is an essential amino acid for infants, but apparently not for adults. • Arginine is produced in the body but not in sufficient quantities to meet protein demand. 16 • Complete or Adequate Proteins: supply all of the essential amino acids. (Animal Proteins) • Incomplete Proteins: Low in one or more of the essential amino acids (Vegetable Proteins) Animal Proteins Source Type of Protein Missing AA Egg Complete None Milk(Dairy) Complete None Meat, fish Complete None 17 Source Wheat Corn Vegetable Proteins Type of Protein Incomplete Incomplete Rice Beans Incomplete Incomplete Peas Quinoa Hemp Incomplete Complete Complete Missing AA Lysine Lysine & Tryptophan Lysine Methionine Tryptophan Methionine 18 • A complete assortment of amino acids can be obtained from a vegetable diet by pairing a vegetable protein missing one essential amino acid with a vegetable that contains it. • The two vegetable proteins are called complementary proteins. – Ex. Rice and Beans 19 Amphoteric Properties of Amino Acids • Amphoteric substances act as acids or bases. – They are acids when they donate protons. – They are bases when they accept protons. • Amino acids can act as acids or bases. – When placed in an acidic solution (low pH), they act as bases by accepting protons and becoming positively charged. – In basic solutions (high pH), they act as acids by donating protons and becoming negatively charged. 20 ALANINE + CH3 NH3 CH O C - O H+ Acid Solution CH3 + NH3 CH CH3 O NH2 CH C NET CHARGE +1 Basic Solution OH- O C OH - O NET CHARGE -1 21 • Amino Acids function as buffers because they can neutralize small increases of acid or base. • Proteins are one of the major buffering systems in the body. 22 ISOELECTRIC POINT (pI) • A Zwitterion, which is electrically neutral overall, can only exist at a specific pH value. • This pH value, called the isoelectric point, is different for each amino acid. • Amino acids with hydrocarbon R groups attain their isoelectric point between pH 5.0 and 7.0 • ex. Leucine pH = 6.0 • Basic amino acids need high pH values to reach their isoelectric points. • ex. Arginine pH = 10.8 23 • Acidic amino acids need low pH values. • ex. Aspartic acid pH = 3.0 • Proteins also have isoelectric points depending on the amino acids that make them up. – At their pH, proteins become insoluble in water, clump together, and precipitate out of solution. 24 The peptide bond Proteins are polymers made up of amino acids. Peptide bond - how the amino acids are linked together to make a protein. H | H2NCCOOH + | R H | H2NCCOOH | R’ H O H | || | H2N - C - C - N - C - COOH | | | R H R’ This is a condensation reaction: H2O is eliminated. 25 • This bond between the two amino acids is called a peptide bond. • Two amino acids joined like this give what is called a dipeptide. CH3 O NH2 CH C Alanine H OH + O NH CH C H Glycine CH3 O -H2O H O NH2CH C NHCH C OH OH alanylglycine (ala-gly) These 2 amino acids could also link the other way. 26 • Any two amino acids can be joined in a similar manner to form dipeptides. • It doesn’t end here ! Each dipeptide still has a COOH and an NH2 that can form new peptide bonds. • Adding a 3rd amino acid gives us a tripeptide. • This process can be continued to get a tetrapeptide, a pentapeptide, and so on until we have a chain of hundreds or even thousands of amino acids. 27 • The chains of amino acids are the proteins. • The shorter chains are often called polypeptides. – Ex. Glucagon with 21 amino acids is a large polypeptide. – Insulin with 51 amino acids is a very small protein. • We will consider a protein to be a peptide chain with a minimum of 30 amino acids. 28 Primary structure of proteins • Primary Structure: What are the amino acids that make up the protein and how are they arranged in the chain ? (The amino acid sequence) ala gly pro arg his ser asn ile thr asp leu trp cys lys tyr gln met glu phe val 29 • The amino acids in a chain are often referred to as residues. – Ex. Ala-gly-lys 3 residue amino acids • The amino acid residue with the free COOH group is called the C-terminal, and the amino acid residue with the free NH2 group is called the N-terminal. • Peptide and protein chains are always written with the N-terminal residue on the left. 30 Peptides N-terminal residue C-terminal residue H O H O H | || | || | H2N - C - C - NH - C - C - N - C - COOH | | | | R R’ H R’’ peptide linkages 31 • The continuing pattern of peptide linkages is called the backbone of the protein molecule. R NHCH O C R' NHCH O R" O C NHCH C The R groups are called the side chains. The 20 different amino acid side chains provide variety and determine the chemical and physical properties. 32 • Each peptide and protein molecule in biological organisms has a different sequence of amino acids. • It is this sequence that allows the protein to carry out its function, whatever it might be. • The number of different protein possibilities is staggering. – Ex. A tripeptide can have 20 different amino acids at each position. • 20 x 20 x 20 = 8000 possible tripeptides 33 • A typical protein with 60 amino acid residues can have up to 2060 different arrangements. • This means that there would be 1 x 1078 possibilities. • 1,000,000,000,000,000,000,000,000,000,000,000, 000,000,000,000,000,000,000,000,000,000,000,0 00,000, 000,000. 34 Secondary structure of proteins Long chains of amino acids will commonly fold or curl into a regular repeating structure. Structure is a result of hydrogen bonding between amino acids within the protein. Common secondary structures are: - helix - pleated sheet Secondary structure adds new properties to a protein like strength, flexibility, ... 35 -Helix One common type of secondary structure. Properties of -helix include strength and low solubility in water. Originally proposed by Pauling and Corey in 1951. 36 -Helix C || O H | N C || OH | HN | N H | N C H || | O N H | N C || O C || O H | C H || H N C | O | || N C O N || C O || O Every amide hydrogen and carbonyl oxygen is involved in a hydrogen bond. There are 3.6 amino acids in each turn. Multiple strands may entwine to make a protofibril. The R groups extend out from the helical portion of the -helix 37 -Helix example myosin head myosin tail ATP and actin binding sites thick filament thin filament actin troponin myosin/actin structure Proteins used in muscle 38 -Pleated sheets Another secondary structure for protein. Held together by hydrogen bonding between adjacent sheets of protein. C | R C | R H | N C || O R | C C || O N | H R H | | C N O || C O || C H | N C | R N | C H | R R | C C || O H | N C || O N | H R | O C || C O || C C | R N | C H | R 39 -Pleated sheets Silk fibroin - main protein of silk is an example of a pleated sheet structure. Composed primarily of glycine and alanine. Stack like corrugated cardboard for extra strength. 40 Linus Pauling http://www.youtube.com/watch?v=yh9Cr5n21EE 41 Collagen Family of related proteins. About one third of all protein in humans. Structural protein Provides strength to bones, tendon, skin, blood vessels. Forms triple helix - tropocollagen. 42 •As an animal grows older, the extent of cross-linking increases and the meat gets tougher. •Treatment with boiling water converts collagen to gelatin. Therefore, cooking meat converts part of the tough connective tissue to gelatin, making the meat more tender. (ex. Stewing chickens) 43 Tanning hides increases the degree of crosslinking, converting skin to leather. 44 • Wool, hair and muscle are all formed from strands of alpha helixes. • These proteins can be stretched because the hydrogen bonds can be elongated and then return to the original configuration. • This is especially true for wool. 45 DISULFIDE BRIDGES • Disulfide bridges are covalent bonds formed when 2 cysteine units are oxidized to form a cystine unit. SH SH oxidation reduction S S The strength of this bond is much greater than that of a hydrogen bond. 46 Fibrous proteins • insoluble in water • form used by connective tissues • silk, collagen, -keratins Globular proteins • soluble in water • form used by cell proteins • 3-D structure - tertiary 47 Tertiary structure of proteins • This refers to how the molecule is folded. It makes the molecule very compact. • Results from interaction of side chains. • Protein folds into a tertiary structure. • This is typical of proteins called globular. – Found in egg and serum albumin, hemoglobin and myoglobin, and enzymes and antibodies. 48 Types of tertiary bonding Possible side chain interactions: - Similar solubilities - Ionic attractions - Attraction between + and - sidechains - Covalent bonding 49 Tertiary structure of proteins Sulfide Crosslink Hydrophobic interaction -S-S- -COO- H3N+- Salt bridge Hydrogen bonding Side chain interactions Help maintain specific structure. Oxidation of cysteine - crosslink formation. O || HO-C-CH-CH2-SH | NH2 oxidation [O] O || HS-CH2-CH-C-OH | NH2 covalent disulfide bond O O || || HO-C-CH-CH2-S - S-CH2-CH-C-OH +H2O | | NH2 NH2 51 Hydrophobic attractions Attractions between R groups of non-polar amino acids. Hydrogen bonding Interaction between polar amino acid R groups. Ionic bonding Bonding between oppositely charged amino acid R groups. 52 CH 110 homework #13 & #14 due Today! CH 110 homework #15 & #16 due Saturday, March 19th Final Exam March 19th 9:00amnoon. 53 Quaternary structure of proteins Many proteins are not single peptide strands. They are combinations of several proteins - aggregate of smaller globular proteins. Conjugated protein - incorporate another type of group that performs a specific function. - prosthetic group 54 Quaternary structure of proteins Aggregate structure This example shows four different proteins and two prosthetic groups. 55 Hemoglobin and myoglobin Hemoglobin oxygen transport protein of red blood cells. Myoglobin oxygen storage protein of skeletal muscles. Both proteins rely on the heme group as the binding site for oxygen. 56 Myoglobin Heme 57 Hemoglobin 2 chains 4 heme 2 chains 58 Hemoglobin and oxygen transport In the lungs, there is an abundance of O2 so oxygen is picked up by the hemoglobin. Hb + 4 O2 Hb(O2)4 When blood reaches the cells, there is a lack of O2 so oxygen is given up by the hemoglobin. Hb + 4 O2 Hb(O2)4 59 Sickle cell anemia Defective gene results in production of mutant hemoglobin - one misplaced amino acid. Glutamate is replaced by valine at 2 of the 547 positions. Still transports oxygen but results in deformed blood cells - elongated, sickle shaped. Difficult to pass through capillaries. Causes organ damage, reduced circulation. Affects 0.4 % of American blacks. 60 Sickled cells 62 63 Comparison of normal and sickle cell hemoglobin Normal Sickle 64 Summary of protein structure primary secondary H O H O H | || | || | H2N - C - C- NH - C - C - N - C - COOH | | | | R R’ H R’’ tertiary quaternary 65 Effect of temperature and pH on proteins • Both will alter the 3-D shape of a protein if you go beyond a ‘normal’ range. • Disorganized protein will no longer act as intended - denatured. They become biologically inactive. • They will clump together - coagulate. – Examples frying an egg HCl in stomach 66 Denaturing of a protein denatured heat or acid coagulated heat or acid 67 •Changes in pH or temperature may not break any of the peptide bonds. •The primary structure is maintained. •If denaturation occurs under extremely mild conditions, the protein may be restored to its original shape. 68 COAGULATION: •Most changes are so drastic that the protein remains denatured. •Protein strands become insoluble and precipitate out of solution (coagulate). •Effects of coagulation are irreversible. 69 Hydrolysis • Will result in protein being reduced to simpler peptides and amino acids. • Amount of hydrolysis depends on pH, time and temperature. O O || || H2N - CH - C - NH - CH - C - OH + H2O | | R R’ H+ or OH- O || H2N - CH - C - OH | R O || H2N - CH - C - OH | R’ 70 • The effect of acid or base is to add or remove H+ to ionic side groups. – Salt bridges and hydrogen bonds are disrupted. • When a protein is placed in a strong acid or base, coagulation may also occur. – Ex. Cheese made from acid coagulated protein (casein) which forms curds. 71 • Tannic acids in burn ointments cause protein coagulation at the site of the burn. • This forms a protective coating which acts as a barrier to further loss of fluids. • A household source of tannic acid is Tea. – Applying dampened tea bags to a burn will precipitate protein and form a protective barrier over the wound. 72 Heat and UV Light • Optimum temperature for most proteins is 37C. • Very few proteins remain biologically active above 50 C. (Some bacteria have protein that remains stable up to 70 C and higher) – Increased Thermal activity (heat or UV) disrupts some of the hydrogen bonds and attractions between non-polar side groups that maintain secondary and tertiary structures. 73 • When you cook food, you are denaturing protein. – Ex. Boiling an egg, frying a steak. • High temperatures are used to disinfect surgical instruments, gowns, and gloves. – AUTOCLAVE. 74 ORGANIC SOLVENTS • Solvents like Ethanol, isopropyl alcohol, and acetone disrupt the hydrogen bonding of proteins by forming their own hydrogen bonds with the protein. • These solvents are used as disinfectants. – A 70% solution of ethanol or IPA can pass thru cell walls of bacteria – Once inside, they cause coagulation of the bacterial proteins within the cell. • A 90% solution isn’t nearly as effective. Why? 75 Heavy Metal Ions • Metal ions like Ag+, Pb 2+, Hg 2+, etc.. cause denaturation of protein. • The heavy metals react with the disulfide bonds and the carboxyl groups of acidic amino acids. • The denatured protein is insoluble and precipitates out of solution. – Ex. 1% AgNO3 placed in eyes of newborn to kill the bacteria that causes gonorrhea. 76 Colloidal silver 77 Other Methods of Denaturation • Agitation: Violent whipping action causes a stretching of globular proteins which turns egg whites into meringues and whipping creams into toppings. How does cream of tartar work? – Which would be best for beating eggs into meringue: a glass bowl or a copper bowl? – Why can canned pineapple be used in gelatin deserts while fresh pineapple can’t be used? 78 Protein gallery Human growth hormone 596 residues Originally obtained from human cadavers. It would cost $20,000 per year to treat one child. Now produced by genetically engineered bacteria. 79 Protein gallery Immunoglobin FC - 262 residuals A ‘Y” shaped protein actually composed of 4 protein chains linked by disulfide bonds. antigen-binding site 80 Protein gallery Lipoprotein 116 residues 2 helical strands 81