Chapter 3 The Same 20 Amino Acids Serve as Building Blocks for All

Proteins in Nature

Homework 2 part 1: Ch. 5 problems 2, 9, 12, 16.

1.1 Almost all chemical reactions occurring in living organisms are catalyzed by enzymes.

1.1.1 Many thousands of enzymes have been discovered, each catalyzing a different kind of chemical reaction.

1.1.2 Life would not occur without enzyme catalysis.

1.1.3 Enzymes are the most varied and most highly specialized proteins.

1. Proteins are extremely versatile in function and crucial in virtually all biological processes.

1.2 Many small molecules and ions are transported by specific proteins.

1.2.1 Some proteins transport molecules from one organ to another, many exist in the blood plasma, e.g., hemoglobin (oxygen), serum albumin (fatty acids), lipoproteins (lipids), and transferrin (iron).

1.2.2 Some proteins transport molecules across plasma membrane or organelle membranes (glucose, amino acids, nucleotides, chloride, potassium, sodium ions but not water).

1.3 Some proteins function as nutrient or storage proteins. For example, many of such kind exist in plant seeds, animal eggs (Ovalbumin of egg white, casein of milk). Ferritin in animal tissue function to store ion (about 4500 ions are stored in the cavity of each ferritin complex!).

1.4 Some proteins are responsible for the coordinated motions (contraction, changing shape, moving about). For example, actin and myosin in muscle and nonmuscle cells. Tubulin and dynein in eukaryotic flagella and cilia.

1.5 Some proteins are responsible for mechanical support (strength and protection)

1.5.1 Collagen in tendons (筋腱 ), cartilage, and leather.

1.5.2 Elastin in ligaments (韧带 ).

1.5.3 Keratin in hair, fingernails, feathers.

1.5.4 Fibroin in silk fibers and spider webs.

1.5.5 Resilin in wing hinges of some insects.

1.6 Some proteins function to defend the organisms

1.6.1 Immunoglobins in vertebrates

1.6.2 Fibrinogen and thrombin in blood-clotting

1.6.3 Snake venom, bacterial toxins, and toxic plant proteins (ricin).

1.7 Some proteins help regulate cellular or physiological activity

1.7.1 Growth factors and transcription factors regulate cell growth and differentiation

1.7.2 Hormones (e.g., insulin, growth hormon) coordinate activities of different cells in multicellular organisms.

1.8 Some proteins are responsible for the generation and transmission of nerve impulses.

1.8.1 Rhodopsin responses to light to generate vision

1.8.2 Acetylcholine receptor are responsible for transmitting nerve impulses at synapses

1.9 Many proteins have other functions. Antifreeze proteins protect the blood of Antarctic fish from freezing. Special heat stable proteins in thermophile bacteria.

Chymotrypsin with its active site

Fireflies emit light catalyzed by luciferase with ATP

Erythrocytes contain a large amount of hemoglobins, the oxygen-transporting protein.

The protein keratin is the chief structural components of hair, scales, horn, wool, nails and feathers.

2. All natural proteins were found to be built from a repertoire of 20 standard -amino acids

2.1 The earliest studies of proteins focused on the free amino acids derived from these proteins.

2.1.1 The 1st amino acid (asparagine) was discovered in 1806 from asparagus (a green vegetable).

2.1.2 The last (threonine) was not identified until 1938!

2.1.3 All the amino acids were given a trivial (common) name. Glutamate from wheat gluten (sticky). Tyrosine from cheese (“tyros” in Greek).

2.2 The 20 -amino acids share common structural features.

2.2.1 Each has a carboxyl group and an amino group (but one has an imino group in proline) bonded to the same carbon atom, designated as the -carbon.

2.2.2 Each has a different side chain (or R group, R=“Remainder of the molecule”).

2.2.3 The -carbons for 19 of them are asymmetric (or chiral), thus being able to have two enantiomers. Glycine has no chirality.

In protein chemistry, we use Greek letter nomenclature.

2.3 The two enantiomers of each amino acid defined by the -carbon are designated D- and L- forms (D for Dextrorotary, L for Levorotary)

2.3.1 The D- and L-forms of amino acids are named in reference to the absolute configuration of D- and L- glyceraldehydes (whose structure was orignally assumed and confirmed by X-ray crystallography later).

2.3.2 Only the L-amino acids have been found in proteins (D-isomers have been found only in small peptides of bacteria cell walls and in some peptide antibiotics).

2.3.3 The correlation of structure (or configuration) with optical rotation is very complex and has not been successful to date! (i.e., the D- and L-signs do not tell anything about their optical rotation!)

2.4 The amino acids ionize in aqueous solutions.

2.4.1 Crystalline amino acids (in neutral aqueous solutions) have melting points much higher than those of other organic molecules of similar size.

2.4.2 The amino acids ionize to various states depending on pH values.

2.4.3 The amino acids (of neutral side chains) exist predominantly as dipolar ions, known as zwitterions (German for “hybrid ions”).

2.5 Each amino acid is given a three-letter abbreviation and a one-letter symbol. They often the first three letter and the first letter. When there is confusion, an alternative is used. They must be remembered. (fig.)

2.6 All proteins in all species (from bacteria to human) are constructed from the same set of 20 amino acids.

2.6.1 All proteins, no matter how different they are in structure and function, are made from the 20 standard amino acids.

2.6.2 This fundamental alphabet of the protein language is at least two billion years old.

Stryer’s method: walk from the amino group to the carboxylgroup, the hydrogen atom is on your left. L-Alanine

Align carbon atoms with L-glyceraldehyde, the amino groupis on the left. Fig. 5-4

The horizontal bonds project out of the plane of the paper,the vertical behind.

Lined up by similarity: chiral to chiral, COO to CHO

Gly, GAla, AVal, VLeu, LMet, MIle, I

Phe, F; Tyr, Y; Trp, W

Ser, SThr, TCys, CPro, PAsn, NGln, Q

Lys, K; Arg, R; His, H

Asp, D; Glu, E

3. The 20 amino acids are usually grouped according to the properties (mainly polarity) of their R groups

3.1 Six amino acids have nonpolar, aliphatic (hydrophobic) R groups.

3.1.1 They are Gly, Ala, Val, Leu, Ile, and Pro.

3.1.2 Gly has a hydrogen as its R group, having minimal steric hindrance.

3.1.3 Pro has an imino group, instead of an amino group, forming a five-membered ring structure, being rigid in conformation.

3.1.4 Pro is often found in the bends of folded protein chains and often present on the surface of proteins.

3.1.5 In protein structure Gly offers the most flexibility, while Pro the least!

3.1.6 Ala, Val, Leu, and Ile, have hydrocarbon R groups, often involved in hydrophobic interactions.

3.2 Phe, Tyr, and Trp have aromatic R groups3.2.1 Phe and Tyr both have benzene rings.3.2.2 Tryptophan has an indole ring.3.2.3 All three participate in hydrophobic intera

ctions.3.2.4 The -OH group in Tyr is an important func

tional group in proteins. (phosphorylation, hydrogen bond, etc)

3.2.5 They are jointly responsible for the light absorption of proteins at 280 nm

A=Log Io/I = cl Lambert-Beer’s law extinction coefficient; c, concentration; l, optical length

3.3 Ser, Thr, Asn, Gln, Cys, and Met have polar, uncharged R groups.

3.3.1 The R groups are more hydrophilic, due to the presence of hydroxyl groups, sulfur atoms, or amide groups.

3.3.2 -SH group of two Cys in proteins can be oxidized to form a covalent disulfide bond.

3.3.3 Cys and Met often participate in hydrophobic interactions.

3.4 Asp and Glu have carboxyl in their R groups. They have net negative charge at pH 7.0, thus usually named as aspartate and glutamate (conjugate base names, instead of aspartic acid and glutamic acid, un-ionized form).

3.5 Arg, Lys, and His have positively charged R groups at pH 7.0.

3.5.1 Their R groups contain guanidino, amino, imidazole groups respectively.

3.5.2 The side chain of His can be positively or uncharged depending on the local environment near pH 7.0

3.6 The extent of hydrophobicity and hydrophilicity of the side chains is reflected by their hydropathy index values. “-” values usually mean hydrophilic, “+” values hydrophobic.

3.7 Nonstandard amino acids are found in certain proteins, generally as a result of post-translational modifications.

3.7.1 These modifications are made after the standard amino acids have been incorporated into proteins.

3.7.2 4-Hydroxyglutamate and 5-hydroxylysine in collagen.

3.7.3 -carboxyglutamate is found in the blood-clotting prothrombin (an enzyme).

3.7.4 Desmosine is a covalent linkage made from four Lys side chains in elastin.

3.7.5 Selenocysteine is found in many enzymes (having been recognized as the 21st amino acid in ribosome-mediated protein synthesis!).

3.7.6 Many additional nonstandard amino acids are found in cells, but not in proteins (e.g., ornithine and citrulline, intermediates in amino acid metabolism).

4. Amino acids, being both weak acids and bases, have characteristic titration curves and pKa values

4.1 Amino acids can act both as acids and bases. The zwitterion form of amino acids are ampholytes. Amino acids can be diprotic and triprotic acids.

4.2 Monoamino monocarboxylic -amino acids (e.g., Gly, Ser, Phe with no ionizable groups) all have similar two-stage titration curves.

4.2.1 The first stage reflects the deprotonation of the -COOH group (pK1).

4.2.2 The second stage reflects the deprotonation of the -NH 3 + group (pK2).

4.2.3 The pKa value of the -COOH is more than 2.0 units smaller than that of acetic acid (pKa of 4.76), that is, a stronger (weak) acid.

4.2.4 These amino acids have two buffering power regions.

4.3 Acidic and basic amino acids have three-stage titration curves. The additional stage is for the ionizable group on the side chains (pKR).

4.4 There is a specific pH (designated pI) at which an amino acid has equal positive and negative charge.

4.4.1 An amino acid does not move in an electric field at its pI, called isoelectric point.

4.4.2 The pI of monoamino monocarboxylic amino acids reflects a status at which the -COOH group is fully deprotonated, but the -NH3+ group has not yet started deprotonating

pI = (pK1+pK2)/2

4.4.3 The pI point of an acidic amino acid reflects a status at which the -COOH is fully deprotonated, but the side chain -COOH and the a-NH3+ group have not yet started deprotonating

pI = (pK1+pKR)/2

4.4.4 The pI point of a basic amino acid reflects a status at which the -COOH and the side chain -NH3+ or -NH+= group have fully deprotonated, but the -NH3+ group not yet deprotonated

pI = (pKR+pK2)/2

4.4.5 The amino acids are positively charged at pH smaller than their pI values, negatively charged at pH larger than their pI values.

An acidic amino acid pI=(pK1+pKR)/2

A basic amino acid pI=(pKR+pK2)/2

5. The 20 amino acids can be separated from each other by ion-exchange chromatography

5.1 Each of the amino acids has a different pI value. Therefore, each amino acid has a different net charge at a given pH.

5.2 The variously charged amino acids bind to charged synthetic resins with various affinities.

5.2.1 When the resin is positively charged, negatively charged amino acids (or other anions) will bind, and vice versa.

5.2.2 Amino acids having the same charge as the resin will not bind.

5.2.3 The positively charged resin is called anion-exchange resin. The negatively charged called cation-exchange resin (e.g., the sulfonated polystyrene).

5.2.4 The resin (serving as the stationary phase) is usually packed in a column (providing the mechanical support and stable fluid flow).

5.3 The bound amino acids can be eluted by running a pH or salt gradient (serving as the mobile phase).

5.3.1 Amino acids will be eluted out in the order of their binding affinity (strongly bound ones being eluted out later).

5.3.2 This way of separating amino acids (or other charged biomolecules) is called ion-exchange chromatography.

5.3.3 Chromatography is a method of separating substances by allowing them to partition between two phases, one mobile, one stationary (differences in charge, size, hydrophobic interactions, specific interactions can be exploited for substance separation with chromatography).

A.J. Martin and R.L. Synge won the Nobel Prize in 1952 for inventing chromatography.

5.4 The mobile phase can percolate through the column at low pressure or high pressure.

5.4.1 To operate under high pressure, specially designed resins and apparatus (the pumps and the plumbing system) are needed.

5.4.2 Using high pressure allows better separation in a much shorter period of time, thus named High Performance Liquid Chromatography (HPLC).

5.5 Quantitative estimates of each amino acid in a mixture can be efficiently carried out using a fully automated Amino Acid Analyzer.

5.5.1 Addition of sample mixture, elution of each amino acid, collection and analysis of fractions, and data recording are all fully automated!

5.5.2 HPLC (ion exchange) column is used in the machine.

Cation-exchangecolumn

Gel filtrationcolumn

Affinitychromatography

6. The amino acids can be detected using various chemical reagents

6.1 The free -amino group of any amino acids will react with ninhydrin to form a purple product.

6.1.1 Detects amino acids nonspecifically.

6.1.2 The imino group of Pro gives a yellow color.

6.1.3 The concentration/amount (micro gram) of amino acid can be determined by measuring optical absorbance (at 440 nm for purple or 550 nm for yellow).

6.2 Other reagents also react with the -amino group, keeping the R group part of the products

6.2.1 1-Fluoro-2,4-dinitrobenzene and dabsyl chloride react to form colored derivatives that are stable under harsh conditions (heating in 6N HCl at 110C for 24 hours!)

6.2.2 Fluorescent derivatives, permitting the detection of nanogram amount of amino acids.

6.2.3 Identity of amino acids can be revealed by comparing with a standard.

7. Amino acids covalently join one another to form peptides

7.1 The -carboxyl group of one amino acid joins with the -amino group of another amino acid by a peptide bond (actually an amide bond)

7.1.1 This is a condensation reaction where a water molecule is liberated or eliminated.

7.1.2 G of the condensation reaction is about 5 kcal/mol, not being able to occur spontaneously (an endergonic reaction).

7.1.3 The condensation reaction can occur repeatedly to form oligopeptides (with less than 50 aa), polypeptides (bwt 50-100 aa), and proteins (longer).

7.2 The peptide chain is directional.

7.2.1 An amino acid unit in a peptide chain is called a residue.

7.2.2 The end having a free -amino group is called amino-terminal or N-terminal.

7.2.3 The end having a free -carboxyl group is called carboxyl-terminal or C-terminal.

7.2.4 By convention, the N-terminal is taken as the beginning of the peptide chain, and put at the left (C-terminal at the right). Biosynthesis starts from the N-terminal.

7.2.5 The peptide chain consist of a regularly repeating main chain (or backbone) and the variable side chains of the residues.

7.2.6 Amino acid residues have an order or sequence on a peptide.

7.2.7 The 20 amino acids are analogous to the 26 letters in English; the number of different peptides made of them is unlimited.

7.3 The size of a peptide can be described by its total number of residues (e.g., a pentapeptide, a octapeptide) or relative molecular mass (molecular weight).

7.3.1 The mean molecular weight of an amino acid residue in a peptide is ~110 dalton.

7.3.2 Most natural polypeptide chains contain between 50 and 2000 amino acid residues, thus having relative molecular mass between 5500 daltons and 220,000 daltons, or 5.5 kDa and 220 kDa, respectively.

7.4 Each peptide has a characteristic titration curve and a isoelectric point (pI).

7.4.1 The titration curve of a peptide reflect the collective behavior of all the acid-base groups.

7.4.2 The peptide would not move in an electric field at its pI. (determined by IEF, isoelectric focusing gel).

7.4.3 2D gel in proteomics: one dimension is IEF separation by pI; the other SDS-PAGE separation by molecular weight.

8. Many short peptides have important biological activities

8.1 Some short peptides (neuropeptides) act as neurotransmitters, neurohormones, and neuromodulators.

8.1.1 These peptides are secreted by the neurons.

8.1.2 The LHRH-like decapeptide (10-residue) act as a neurotransmitter for the frog sympathetic ganglia.

8.1.3 Thyrotropin-releasing factor (3-residue) is formed in the hypothalamus and stimulates the release of thyrotropin from the anterior pituitary gland.

8.1.4 Oxytocin (9-residue) is secreted by the posterior pituitary and stimulates uterine contraction.

8.1.5 The opioid peptides (including mainly enkaphalins, endorphins, and dynorphins) have been implicated in the control of pain, responses to stress, and other functions.

8.1.6 Some drugs, like morphine and heroin, generate their addictive effect by binding to opioid peptide receptors!

8.2 Some short peptides act as antibiotics

8.2.1 Gramicidin A (15-residue) is a well studied peptide antibiotic (from Bacillus brevis). Its structure has been determined.

8.2.2 It contains alternating L- and D-amino acid residues.

8.2.3 It is not synthesized on ribosomes!

8.2.4 Gramicidin S (10-residue, circular) is another antibiotic also from Bacillus brevis.

8.2.5 Peptide antibiotics have also been found in frog skins, neutrophile cells, and insects.

8.3 Many short peptides are used as defensive poisons.

8.3.1 -Amantin (8-residue, circular) in mushroom is an extremely toxic peptide (inhibiting RNA polymerases II and III at picomolar levels!)

8.3.2 Very toxic short peptides are also found in snake venom, spider.

8.4 Many vertebrate hormones are small polypeptides.

8.4.1 Insulin (51-residue) is produced by the pancreas and acts to lower blood glucose level, after food intake.

8.4.2 Glucagon (29-residue) is also produced by the pancreas and acts to increase the blood glucose level.

8.4.3 Corticotropin (or adrenocorticotropin, 39-residue) is produced by the anterior pituitary gland and stimulates the growth of adrenal cortex and secretion of corticosteroid.

8.4.4 Vasopressin (9-residue) stimulates the reabsorption of water in the distal tubules of the kidney (diabetes insipidus patients have deficient vasopression)

8.5 Many such bioactive peptides are present in exceedingly small amounts (thus difficult to discover!) and acts at very low concentrations.

Bioactive short peptides can be selected by making random peptide libraries (through chemical synthesis, combinatory chemistry, or phage display). Very little is known about the receptors of these bioactive peptides. They should be good potential drug targets.

9. Peptides can be synthesized chemically

9.1 Peptides of up to 150 residues can be synthesized by automated solid-phase methods mainly invented by R. Bruce Merrifield (who won the 1984 Nobel Prize in Chemistry for this).

9.1.1 Amino acids are added stepwise to a growing peptide chain that is linked to an insoluble matrix, such as polystyrene beads.

9.1.2 A major advantage is that the desired product at each stage is bound the insoluble beads with other chemicals easily filtered and washed away.

9.1.3 The synthesis starts with fixing the C-terminal amino acid on the insoluble beads through its -carboxyl group. This is in the reverse direction of biosynthesis.

9.1.4 The -amino group of the next amino acid to be added is protected and its carboxyl group activated. The amino group is protected by the t-butyloxycarbonyl group (t-boc) and deprotected by CF3COOH. The carboxyl group is activated by dicyclohexylcarbodiimide (DCC).

9.1.5 The peptide bond is formed by the free -amino group (deprotected) of the fixed C-terminal residue attacking the DCC activated -carboxyl group of the free amino acid in solution.

9.1.6 After washing away unreacted free amino acids and other reagents, step 9.1.4 and 9.1.5 are repeated. The synthesized peptide is eventually cleaved off from the resin by adding HF.

9.2 The efficiency of this solid phase synthesis is much lower than biosynthesis in living organisms.

9.2.1 Synthesizing a 100 amino acid peptide will take about 4 days to finish with a fully automated machine with reasonable yield.

9.2.2 The same peptide would be synthesized with exquisite fidelity in about 5 seconds in a bacterial cell!

9.3 Peptides having natural activity have been synthesized chemically.

9.3.1 The complete bovine insulin was first synthesized and show to be the same as the natural insulin in China in 1965!

9.3.2 Merrifield also synthesized the interferon (155 aa) and ribonuclease (124 aa).