Amino Acids, Peptides, and Proteins

Amino Acids, Peptides, and Proteins
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Objectives Draw a general amino acid and identify the two functional groups common to all. Classify each amino acid according to the chemical nature of its R group. Define the meaning of an essential amino acid. Draw the reaction that joins two amino acids to form a peptide bond. Describe and differentiate primary, secondary, tertiary, and quaternary protein structures. Describe and differentiate co-enzymes and prosthetic groups. List and discuss four forces that stabilize globular protein structure. List important structural similarities and differences between myoglobin and hemoglobin. Describe the mutation present in hemoglobin giving rise to sickle cell disease.

What is an amino acid? Twenty different kinds of amino acids are used by living organisms to produce proteins An amino acid is a molecule containing an amine (-NH2) an acid (-COOH) and a third chemical group (-R) that defines the amino acid. In glycine, the simplest amino acid, R is –H, or a hydrogen atom. In alanine, R = -CH3. The R groups give specific properties to each amino acid, and to the proteins composed of amino acids. R | Structure of an amino acid: H2N – C – COOH H

Fundamentals While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively. They are classified as , , , etc. amino acids according the carbon that bears the nitrogen.

The 20 Key Amino Acids More than 700 amino acids occur naturally, but 20 of them are especially important. These 20 amino acids are the building blocks of proteins. All are -amino acids. They differ in respect to the group attached to the  carbon. These 20 are listed in Table 27.1.

Amino Acids + NH3  – CO2 + – H3NCH2CH2CO2  + – H3NCH2CH2CH2CO2 
an -amino acid that is an intermediate in the biosynthesis of ethylene  a -amino acid that is one of the structural units present in coenzyme A + H3NCH2CH2CO2 –  + H3NCH2CH2CH2CO2 – a -amino acid involved in the transmission of nerve impulses 

Classification of Amino Acids
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C O – R H H3N + The amino acids obtained by hydrolysis of proteins differ in respect to R (the side chain). The properties of the amino acid vary as the structure of R varies.

C O – R H H3N + The major differences among the side chains concern: Size and shape Electronic characteristics

General categories of a-amino acids
nonpolar side chains polar but nonionized side chains acidic side chains basic side chains

Amino Acid R-groups Non-Polar Hydrophobic Polar Charged Uncharged
Tryptophan Phenylalanine Isoleucine Tyrosine Leucine Valine Methionine Polar Charged Arginine (+) Glutamic acid (-) Aspartic Acid (-) Lysine (+) Histidine (+) Uncharged Cysteine Proline Serine Glutamine Asparagine Ambivalent Glycine Threonine Alanine

1. Hydrophobic (non-polar) residues
Usually interior of proteins away from water. Hydrocarbon: do not contain polar atoms.

- - Charged Amino Acids + + + Arginine [Arg] Glutamate [Glu]
Aspartate [Asp] Lysine [Lys] + Histidine [His]

Hydrophobic Indexes Arginine Arg [R] -11.2 Glycine Gly [G] 0
Glutamic Acid Glu [E] -9.9 Aspartic Acid Asp [D] -7.4 Lysine Lys [K] -4.2 Histidine His [H] -3.3 Cysteine Cys [C] -2.8 Proline Pro [P] -0.5 Serine Ser [S] -0.3 Glutamine Gln [Q] -0.3 Asparagine Asn [N] -0.2 Glycine Gly [G] 0 Threonine Thr [T] 0.4 Alanine Ala [A] 0.5 Methionine Met [M] 1.3 Valine Val [V] 1.5 Leucine Leu [L] 1.8 Tyrosine Tyr [Y] 2.3 Isoleucine Ile [I] 2.5 Phenylalanine Phe [F] 2.5 Tryptophan Trp [W] 3.4

Essential amino acids Definition - Those amino acids that cannot be synthesized in the body in sufficient quantities for anabolic needs. In humans, Isoleucine Leucine Valine Tryptophan Methionine Lysine Phenylalanine Threonine Histidine

White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
20 Amino acids Glycine (G) Alanine (A) Valine (V) Isoleucine (I) Leucine (L) Proline (P) Methionine (M) Phenylalanine (F) Tryptophan (W) Asparagine (N) Glutamine (Q) Serine (S) Threonine (T) Tyrosine (Y) Cysteine (C) Asparatic acid (D) Glutamic acid (E) Lysine (K) Arginine (R) Histidine (H) White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

C O – H H3N + Glycine (Gly or G) Glycine is the simplest amino acid. It is the only one in the table that is achiral. In all of the other amino acids in the table the  carbon is a chirality center.

H O + – H3N C C O CH3 Alanine (Ala or A) Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic.

H O + – H3N C C O CH(CH3)2 Valine (Val or V)

H O + – H3N C C O CH2CH(CH3)2 Leucine (Leu or L)

C O – CH3CHCH2CH3 H H3N + Isoleucine (Ile or I)

H O + – H3N C C O CH3SCH2CH2 Methionine (Met or M) The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable.

C O – CH2 H H2N + H2C C H2 Proline (Pro or P) Proline is the only amino acid that contains a secondary amine function. Its side chain is nonpolar and cyclic.

C O – CH2 H H3N + Phenylalanine (Phe or F) The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.

C O – CH2 H H3N + N Tryptophan (Trp or W) The side chain in tryptophan (a nonpolar amino acid) is larger and more polarizable than the benzyl group of phenylalanine.

H O + – H3N C C O CH2OH Serine (Ser or S) The —CH2OH side chain in serine can be involved in hydrogen bonding.

H O + – H3N C C O CH3CHOH Threonine (Thr or T) The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine.

H O + – H3N C C O CH2SH Cysteine (Cys or C) The side chains of two remote cysteines can be joined by forming a covalent S—S bond.

C O – CH2 H H3N + OH Tyrosine (Tyr or Y) The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding.

C O – H H3N + H2NCCH2 Asparagine (Asn or N) The side chains of asparagine and glutamine (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding.

C O – H H3N + H2NCCH2CH2 Glutamine (Gln or Q)

C O – H H3N + OCCH2 Aspartic Acid (Asp or D) Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They are negatively charged and can form ionic bonds with positively charged species.

C O – H H3N + OCCH2CH2 Glutamic Acid (Glu or E)

H O + – H3N C C O Lysine + (Lys or K) CH2CH2CH2CH2NH3 Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are positively charged and can form ionic bonds with negatively charged species.

H O + – Arginine H3N C C O (Arg or R) CH2CH2CH2NHCNH2 + NH2

H O + – H3N C C O Histidine (His or H) Histidine is a basic amino acid, but less basic than lysine and arginine. Histidine can interact with metal ions and can help move protons from one site to another. CH2 NH N

Stereochemistry of Amino Acids
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Configuration of -Amino Acids
Glycine is achiral. All of the other amino acids in proteins have the L-configuration at their carbon. H3N + H R CO2 –

Amino Acids All DNA encoded aa are  All are chiral, except Glycine
R = H All DNA encoded aa are usually L-

Acid-Base Behavior of Amino Acids
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Recall While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3+ and —CO2– respectively. How do we know this?

For aa with basic R-groups, we require higher pHs, and
aa are high melting point solids! Why? Answer = aa are ionic compounds under normal conditions Isoelectric Point = concentration of zwitterion is at a maximum and the concentration of cations and anions is equal For aa with basic R-groups, we require higher pHs, and for aa with acidic R-groups, we require lower pHs to reach the Isoelectric Point

e.g. Isoelectric point for gly pH = 6.0 Asp pH = 3.0 Lys pH = 9.8
Isoelectric Point is the pH at which an aa or peptide carries no net charge. i.e. [RCOO-] = [RNH3+] So, for basic R-groups, we require higher pHs, and for acidic R-groups we require lower pHs e.g. Isoelectric point for gly pH = 6.0 Asp pH = 3.0 Lys pH = 9.8 Arg pH = 10.8

The properties of glycine:
high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent O OH H2NCH2C •• • • – H3NCH2C + more consistent with this than this

called a zwitterion or dipolar ion
Properties of Glycine The properties of glycine: high melting point: (when heated to 233°C it decomposes before it melts) solubility: soluble in water; not soluble in nonpolar solvent more consistent with this called a zwitterion or dipolar ion – • • O H3NCH2C •• +

Acid-Base Properties of Glycine
The zwitterionic structure of glycine also follows from considering its acid-base properties. A good way to think about this is to start with the structure of glycine in strongly acidic solution, say pH = 1. At pH = 1, glycine exists in its protonated form (a monocation). O OH H3NCH2C + •• • •

Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is it the proton of the carboxyl group?" You can choose between them by estimating their respective pKas. typical ammonium ion: pKa ~9 typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • •

The more acidic proton belongs to the CO2H group. It is the first one removed as the pH is raised. typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • •

Therefore, the more stable neutral form of glycine is the zwitterion. – • • O H3NCH2C •• + typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • •

The measured pKa of glycine is 2.34. Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an electron-withdrawing, acid-strengthening substituent on the  carbon. typical carboxylic acid: pKa ~5 O OH H3NCH2C + •• • •

A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further. – • • O H3NCH2C •• + – • • O H2NCH2C •• HO – The pKa for removal of this proton is This value is about the same as that for NH4+ (9.3).

Isoelectric Point pI O OH H3NCH2C +
•• • • The pH at which the concentration of the zwitterion is a maximum is called the isoelectric point. Its numerical value is the average of the two pKas. The pI of glycine is 5.97. pKa = 2.34 – • • O H3NCH2C •• + pKa = 9.60 – • • O H2NCH2C ••

Acid-Base Properties of Amino Acids
One way in which amino acids differ is in respect to their acid-base properties. This is the basis for certain experimental methods for separating and identifying them. Just as important, the difference in acid-base properties among various side chains affects the properties of the proteins that contain them. Table 27.2 gives pKa and pI values for amino acids with neutral side chains.

Amino Acids with Neutral Side Chains
– H H3N + pKa1 = pKa2 = 9.60 pI = 5.97 Glycine

– CH3 H + pKa1 = pKa2 = 9.69 pI = 6.00 Alanine

– CH(CH3)2 H + pKa1 = pKa2 = 9.62 pI = 5.96 Valine

– CH2CH(CH3)2 H + pKa1 = pKa2 = 9.60 pI = 5.98 Leucine

pKa1 = pKa2 = 9.60 pI = 5.98 + – Isoleucine H3N C C O CH3CHCH2CH3

pKa1 = pKa2 = 9.21 pI = 5.74 + – Methionine H3N C C O CH3SCH2CH2

– H + CH2 H2C C H2 pKa1 = pKa2 = pI = 6.30 Proline

– H + CH2 pKa1 = pKa2 = 9.13 pI = 5.48 Phenylalanine

– H + CH2 N pKa1 = pKa2 = 9.39 pI = 5.89 Tryptophan

pKa1 = pKa2 = 8.80 pI = 5.41 + – Asparagine H3N C C O H2NCCH2 O

– H + H2NCCH2CH2 pKa1 = pKa2 = 9.13 pI = 5.65 Glutamine

– CH2OH H + pKa1 = pKa2 = 9.15 pI = 5.68 Serine

pKa1 = pKa2 = 9.10 pI = 5.60 + – Threonine H3N C C O CH3CHOH

– H + CH2 OH pKa1 = pKa2 = 9.11 pI = 5.66 Tyrosine

– CH2SH H + pKa1 = pKa2 = 8.18 pI = 5.07 Cysteine

Amino Acids with Ionizable Side Chains
pKa1 = pKa2 = 3.65 pKa3 = pI = 2.77 + – Aspartic acid H3N C C O OCCH2 O – For amino acids with acidic side chains, pI is the average of pKa1 and pKa2.

pKa1 = pKa2 = 4.25 pKa3 = pI = 3.22 + – Glutamic acid H3N C C O OCCH2CH2 – O

H3N C O – H + CH2CH2CH2CH2NH3 pKa1 = pKa2 = 8.95 pKa3 = pI = 9.74 Lysine For amino acids with basic side chains, pI is the average of pKa2 and pKa3.

H3N C O – H + CH2CH2CH2NHCNH2 NH2 pKa1 = pKa2 = 9.04 pKa3 = pI = 10.76 Arginine

H3N C O – H + CH2 NH N pKa1 = pKa2 = 6.00 pKa3 = pI = 7.59 Histidine

Reactions of Amino Acids
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Acylation of Amino Group
The amino nitrogen of an amino acid can be converted to an amide with the customary acylating agents. O CH3COCCH3 O H3NCH2CO – + + CH3CNHCH2COH O (89-92%)

Esterification of Carboxyl Group
The carboxyl group of an amino acid can be converted to an ester. The following illustrates Fischer esterification of alanine. O H3NCHCO – + CH3 + CH3CH2OH HCl (90-95%) O H3NCHCOCH2CH3 + CH3 – Cl

Ninhydrin Test OH O O H3NCHCO – + R + O N – O RCH + CO2 + H2O +
Amino acids are detected by the formation of a purple color on treatment with ninhydrin. OH O O H3NCHCO – + R + O N – O RCH + CO2 + H2O +

Some Biochemical Reactions of Amino Acids
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Decarboxylation Decarboxylation is a common reaction of -amino acids. An example is the conversion of L-histidine to histamine. Antihistamines act by blocking the action of histamine. CH2CHCO2 – NH3 + N H N

Decarboxylation CH2CH2 NH2 N H N –CO2, enzymes CH2CHCO2 – NH3 + N H N

Neurotransmitters – CO2
OH CO2 – H H3N + The chemistry of the brain and central nervous system is affected by neurotransmitters. Several important neurotransmitters are biosynthesized from L-tyrosine. L-Tyrosine

Neurotransmitters – CO2
The common name of this compound is L-DOPA. It occurs naturally in the brain. It is widely prescribed to reduce the symptoms of Parkinsonism. + H3N H H H HO OH L-3,4-Dihydroxyphenylalanine

Neurotransmitters H Dopamine is formed by decarboxylation of L-DOPA. H2N H H H HO OH Dopamine

Neurotransmitters H H2N H H OH HO OH Norepinephrine

Neurotransmitters H CH3NH H H OH HO OH Epinephrine

Peptides 4

Peptides Peptides are compounds in which an amide bond links the amino group of one -amino acid and the carboxyl group of another. An amide bond of this type is often referred to as a peptide bond.

Peptide Bond Formation
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Peptide bond formation
condensation

Peptide bond formation
Primary Structure

aa are covalently linked by amide bonds (Peptide Bonds)
The resulting molecules are called Peptides & Proteins Features of a Peptide Bond; Usually inert Planar to allow delocalisation Restricted Rotation about the amide bond Rotation of Groups (R and R’) attached to the amide bond is relatively free

Primary Structure is the order (or sequence) of amino acid residues
that are part of a peptide or aa protein are referred to as residues. Peptides are made up of about 50 residues, and do not possess a well-defined 3D-structure Proteins are larger molecules that usually contain at least 50 residues, and sometimes The most important feature of proteins is that they possess well-defined 3D-structure. Primary Structure is the order (or sequence) of amino acid residues Peptides are always written and named with the amino terminus on the left and the carboxy terminus on the right

Alanine and Glycine CH3 O C + H – H3N O C H H3N + –

Alanylglycine CH3 O C H3N + H N – Two -amino acids are joined by a peptide bond in alanylglycine. It is a dipeptide.

Alanylglycine CH3 O C H3N + H N – N-terminus C-terminus Ala—Gly AG

Alanylglycine and glycylalanine are constitutional isomers
CH3 O C H3N + H N – Alanylglycine Ala—Gly AG H O C H3N + N CH3 – Glycylalanine Gly—Ala GA

Alanylglycine CH3 O C H3N + H N – The peptide bond is characterized by a planar geometry.

Strong Acid Required to hydrolyse peptide bonds

Cysteine residues create Disulfide Bridges between chains
This does not reveal Primary Structure

Higher Peptides Peptides are classified according to the number of amino acids linked together. dipeptides, tripeptides, tetrapeptides, etc. Leucine enkephalin is an example of a pentapeptide.

Tyr—Gly—Gly—Phe—Leu YGGFL
Leucine Enkephalin Tyr—Gly—Gly—Phe—Leu YGGFL

Oxytocin is a cyclic nonapeptide.
Ile—Gln—Asn Tyr Cys S Cys—Pro—Leu—GlyNH2 1 2 3 4 5 6 7 8 9 C-terminus N-terminus Oxytocin is a cyclic nonapeptide. Instead of having its amino acids linked in an extended chain, two cysteine residues are joined by an S—S bond.

Oxytocin S—S bond An S—S bond between two cysteines is often referred to as a disulfide bridge.

Introduction to Peptide Structure Determination
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Primary Structure The primary structure is the amino acid sequence plus any disulfide links.

Proteins are linear polymers of amino acids
NH3＋ C COOー＋ NH3＋ C COOー＋ H H A carboxylic acid condenses with an amino group with the release of a water H2O H2O R1 R2 R3 NH3＋ C CO NH C CO NH C CO H Peptide bond H Peptide bond H The amino acid sequence is called as primary structure F T D A G S K A N G S

Classical Strategy 1. Determine what amino acids are present and their molar ratios. 2. Cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragments. 3. Identify the N-terminus and C-terminus in the parent peptide and in each fragment. 4. Organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence.

Amino Acid Analysis 4

Amino Acid Analysis Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids. The mixture is separated by ion-exchange chromatography, which depends on the differences in pI among the various amino acids. Amino acids are detected using ninhydrin. Automated method; requires only 10-5 to 10-7 g of peptide.

Partial Hydrolysis of Proteins
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Partial Hydrolysis of Peptides and Proteins
Acid-hydrolysis of the peptide cleaves all of the peptide bonds. Cleaving some, but not all, of the peptide bonds gives smaller fragments. These smaller fragments are then separated and the amino acids present in each fragment determined. Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis.

Partial Hydrolysis of Peptides and Proteins
The enzymes that catalyze the hydrolysis of peptide bonds are called peptidases, proteases, or proteolytic enzymes.

Proteases R2 Pro R1 Pro * * * * *

Trypsin Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine. lysine or arginine NHCHC O R' R" R

Chymotrypsin Chymotrypsin is selective for cleaving the peptide bond to the carboxyl group of amino acids with an aromatic side chain. phenylalanine, tyrosine, tryptophan NHCHC O R' R" R

Carboxypeptidase Carboxypeptidase is selective for cleaving the peptide bond to the C-terminal amino acid. protein H3NCHC O R + NHCHCO – C

End Group Analysis 4

End Group Analysis Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left. We need to know what the N-terminal and C-terminal amino acids are. The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis. Several chemical methods have been developed for identifying the N-terminus. They depend on the fact that the amino N at the terminus is more nucleophilic than any of the amide nitrogens.

Sanger's Method The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene. 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution (Section 23.5). F O2N NO2

Sanger's Method 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. F O2N NO2 NHCH2C NHCHCO CH3 NHCHC CH2C6H5 H2NCHC O CH(CH3)2 – + O2N NO2 NHCH2C NHCHCO CH3 NHCHC CH2C6H5 O CH(CH3)2 –

Sanger's Method Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group. H3O+ O O2N NO2 NHCHCOH CH(CH3)2 H3NCHCO– CH3 + H3NCH2CO– O CH2C6H5 O2N NO2 NHCH2C NHCHCO CH3 NHCHC CH2C6H5 O CH(CH3)2 –

Insulin 4

Insulin Insulin is a polypeptide with 51 amino acids. It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids). The following describes how the amino acid sequence of the B chain was determined.

Primary Structure of Bovine Insulin
N terminus of A chain 5 15 10 20 25 30 S F V N Q H L C G A Y E R I K P T C terminus of A chain N terminus of B chain C terminus of B chain

The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA

FVNQHLCGSHL VGAL VCGERGF YTPKA

Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC

FVNQHLCGSHL SHLV LVGA VGAL ALT TLVC VCGERGF YTPKA

Phenylalanine (F) is the N terminus. Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA Overlaps between the above peptide sequences were found in four additional peptides: SHLV LVGA ALT TLVC Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.

FVNQHLCGSHL SHLV LVGA VGAL ALT TLVC VCGERGF GFFYTPK YTPKA

FVNQHLCGSHL SHLV LVGA VGAL ALT TLVC VCGERGF GFFYTPK YTPKA FVNQHLCGSHLVGALTLVCGERGFFYTPKA

Insulin The sequence of the A chain was determined using the same strategy. Establishing the disulfide links between cysteine residues completed the primary structure.

The Edman Degradation and Automated Sequencing of Peptides
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Edman Degradation 1. Method for determining N-terminal amino acid. 2. Can be done sequentially one residue at a time on the same sample. Usually one can determine the first 20 or so amino acids from the N-terminus by this method. g of sample is sufficient. 4. Has been automated.

Edman Degradation The key reagent in the Edman degradation is phenyl isothiocyanate. N C S

Edman Degradation Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid. peptide H3NCHC O R + NH C6H5N C S +

Edman Degradation peptide C6H5NHCNHCHC O R NH S peptide H3NCHC O R + NH C6H5N C S +

Edman Degradation peptide C6H5NHCNHCHC O R NH S The product is a phenylthiocarbamoyl (PTC) derivative. The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide.

Edman Degradation peptide C6H5NHCNHCHC O R NH S HCl C6H5NH C S N CH R O peptide H3N + +

Edman Degradation The product is a thiazolone. Under the conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH) derivative. C6H5NH C S N CH R O peptide H3N + +

Edman Degradation C N HN CH R O S C6H5 The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation. C6H5NH C S N CH R O peptide H3N + +

Secondary Structures of Peptides and Proteins
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Levels of Protein Structure
Primary structure = the amino acid sequence plus disulfide links Secondary structure = conformational relationship between nearest neighbor amino acids  helix pleated  sheet

Levels of Protein Structure
The -helix and pleated  sheet are both characterized by: planar geometry of peptide bond anti conformation of main chain hydrogen bonds between N—H and O=C

a-helixes Intra-chain H-bonds Secondary Structure

 Helix Shown is an  helix of a protein in which all of the amino acids are L-alanine. Helix is right-handed with 3.6 amino acids per turn. Hydrogen bonds are within a single chain. Protein of muscle (myosin) and wool (-keratin) contain large regions of -helix. Chain can be stretched.

b-strands Inter-chain H-bonds Secondary Structure

Two Types of b-Pleated Sheets

Pleated  Sheet Shown is a  sheet of protein chains composed of alternating glycine and alanine residues. Adjacent chains are antiparallel. Hydrogen bonds between chains. van der Waals forces produce pleated effect.

Tertiary Structure of Peptides and Proteins
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Specific compact structure for one entire polypeptide
Tertiary Structure Specific compact structure for one entire polypeptide Chain stabilizing primarily through weak bonds

Tertiary Structure Refers to overall shape (how the chain is folded) Fibrous proteins (hair, tendons, wool) have elongated shapes Globular proteins are approximately spherical most enzymes are globular proteins an example is carboxypeptidase

Tertiary Structure This is the 3D structure resulting from further regular folding of the polypeptide chains using H-bonding, Van der Waals, disulfide bonds and electrostatic forces – Often detected by X-ray crystallographic methods Globular Proteins – “Spherical Shape” , include Insulin, Hemoglobin, Enzymes, Antibodies ---polar hydrophilic groups are aimed outwards towards water, whereas non-polar “greasy” hydrophobic hydrocarbon portions cluster inside the molecule, so protecting them from the hostile aqueous environment Soluble Proteins Fibrous Proteins – “Long thin fibres” , include Hair, wool, skin, nails – less folded e.g. keratin - the -helix strands are wound into a “superhelix”. The superhelix makes one complete turn for each 35 turns of the -helix.

Bays or pockets in proteins are called Active Sites
In globular proteins this tertiary structure or macromolecular shape determines biological properties Bays or pockets in proteins are called Active Sites Enzymes are Stereospecific and possess Geometric Specificity The range of compounds that an enzyme excepts varies from a particular functional group to a specific compound Emil Fischer formulated the lock-and-key mechanism for enzymes All reactions which occur in living cells are mediated by enzymes and are catalysed by Some enzymes may require the presence of a Cofactor. This may be a metal atom, which is essential for its redox activity. Others may require the presence of an organic molecule, such as NAD+, called a Coenzyme. If the Cofactor is permanently bound to the enzyme, it is called a Prosthetic Group.

For a protein composed of a single polypeptide molecule, tertiary structure is the highest level of structure that is attained Myoglobin and hemoglobin were the first proteins to be successfully subjected to completely successful X-rays analysis by J. C. Kendrew and Max Perutz (Nobel Prize for Chemistry 1962) Quaternary Structure When multiple sub-units are held together in aggregates by Van der Waals and electrostatic forces (not covalent bonds) Hemoglobin is tetrameric myglobin For example, Hemoglobin has four heme units, the protein globin surrounds the heme – Takes the shape of a giant tetrahedron – Two identical  and  globins. The  and  chains are very similar but distinguishable in both primary structure and folding

Hb monomer (or myoglobin)
Tertiary structure Quaternary structure Hb monomer (or myoglobin) Hb a2b2 tetramer

Carboxypeptidase Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus. It is a metalloenzyme containing Zn2+ at its active site. An amino acid with a positively charged side chain (Arg-145) is near the active site.

Carboxypeptidase tube model ribbon model Disulfide bond Zn2+ Arg-145
N-terminus C-terminus tube model ribbon model

Myoglobin Heme N-terminus C-terminus Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen transport).

Protein Quaternary Structure: Hemoglobin
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Protein Quaternary Structure
Some proteins are assemblies of two or more chains. The way in which these chains are organized is called the quaternary structure. Hemoglobin, for example, consists of 4 subunits. There are 2  chains (identical) and 2  chains (also identical). Each subunit contains one heme and each protein is about the size of myoglobin.

Protein Structure Primary structure is the amino acid sequence.
Secondary structure is how the amino acids in sequence fold up locally. Examples are a-helixes and b-strands and loops. Tertiary structure is the 3-dimensional folding of the secondary structural elements and connecting loops in space. Quaternary structure is the association of multiple subunits, each with a tertiary structure and each a unique gene product.

Stabilization of Protein Structure
Electrostatic interactions involve the interaction of (+) and (-) charged side groups. Hydrogen bonds involve sharing of a hydrogen atom between two eletronegative atoms (e.g., O, N). Van der Waal’s forces are weak forces based on optimal overlap of adjacent electronic orbitals. Can be repulsive. Hydrophobic interactions are, by far, the most powerful force stabilizing protein structure. Basis of force is entropy gain realized by burying hydrophobic residues.

Cofactors Cofactors are exogenous molecules that associate with proteins to yield full activity. In the absence of cofactor, protein is an apoprotein. Co-enzymes are soluble and associate transiently with enzyme during catalytic cycle. An example is vitamin K in activation of blood clotting enzymes. Prosthetic groups are covalently attached to the protein. Examples are heme, in hemoglobin, and riboflavin, in flavoproteins.

Denaturation agents Protein Denaturation
Definition: Disruption of any of the bonds that stabilize the secondary, tertiary or quaternary structure. However, the covalent amide bonds of the primary structure are not affected. Denaturation agents Heat: Break apart hydrogen bonds and disrupt hydrophobic attractions between nonpolar side groups. Acids/Bases: Break hydrogen bonds between polar R groups and disrupt the ionic bonds (salt bridges). Organic Compounds: Ethanol and isopropanol act as disinfectants by forming their own hydrogen bonds with a protein and disrupting the hydrophobic interactions. Heavy metal ions: Heavy metal ions like Ag+, Pb2+ and Hg2+ React with S-S bonds to form solids. Agitation: Stretches chains until bonds break

Different Classification of Proteins
On the basis of: Shape: Globular Fibrilar Homo or hetero Function

Immunoglobulin Structure
Heavy & Light Chains Disulfide bonds Inter-chain Intra-chain CH1 VL CL VH CH2 CH3 Hinge Region Carbohydrate Disulfide bond

Some design principles
Globular Proteins Some design principles Globular proteins fold so as to "bury" the hydrophobic side chains, minimizing their contact with water Most polar residues face the outside of the protein and interact with solvent Most hydrophobic residues face the interior of the protein and interact with each other Packing of residues is close, but protein interiors contain some empty space The empty space is in the form of small cavities

More design principles
Globular Proteins More design principles "Random coil" is not random Structures of globular proteins are not static Various elements and domains of protein move to different degrees Some segments of proteins are very flexible and disordered. Myoglobin and hemoglobin are typical examples of globular proteins. Both are heme-containing proteins and each is involved in oxygen metabolism.

Objectives Diagram and describe the effect of oxygen on the position of iron relative to the heme plane. Describe how cooperative binding of oxygen by hemoglobin improves its effectiveness as an oxygen carrier. Describe the relationship between Hb structure to the Bohr effect and explain its physiological significance.. Discuss how carbon dioxide affects the affinity of Hb for oxygen and why this is physiologically significant. Explain the effect of bisphosphoglycerate (BPG) on the affinity of Hb for oxygen and how this is related to altitude and HbF. Explain how carbon monoxide (CO) binds to Hb and its affinity relative to that of oxygen.. Describe the molecular basis of thalassemias and the aberrant Hb that are produced in these diseases.. List three embryonic forms of Hb..

Myoglobin: 2o and 3o aspects
Myoglobin is a single peptide chain of 153 residues arranged in eight a-helical regions labeled A-H. The heme cofactor is the oxygen binding site so it is necessary for myoglobin’s function, oxygen storage in mammalian muscle tissue. His E7 and F8 are important for binding the heme group within the protein and for stabilizing bound oxygen.

Myoglobin and Hemoglobin
Mb is monomer, Hb is a tetramer (a2b2). Hb subunits are structurally similar to Mb, with 8 a-helical regions, no b-strands and no water. Both contain heme prosthetic group Both Mb and Hb contain proximal and distal histidines. Affinity of Mb for oxygen is high, affinity of Hb for oxygen is low.

Myoglobin &Hemoglobin
Two related protein for O2 transportation. Mb has one chain Hb has four chains Each chain has two parts: a globin ( protein) and a heme ( non-protein)

Myoglobin An O2 transport protein in muscle
A Globin( globular soluble protein), 151 residues that contains 8 a-helices (A,B,C,…..H) Contains a heme prosthetic group Binds heme in hydrophobic pocket. Polar groups exposed to solvent, Non-polar groups buried.

Myoglobin: 2o and 3o structure

The Heme Prosthetic Group
• Protoporphyrin with Fe(II) • Covalent attachment of Fe via His F8 side chain • Additional stabilization via hydrophobic interaction • Fe(II) state is active, Fe(III) [oxidized] • Fe(II) atom in heme binds O2

Binding of O2 to Heme Binding of O2 to a free heme group is irreversible ( heme- heme sandwich) Enclosure in a protein( globin) allows reversible binding O2 has only limited solubility (1 X 10-4 M) in water Solubility problem overcome by binding to proteins • Binding of O2 alters heme structure Bright scarlet color of blood in arteries Dark purple color of blood in veins

O O C C H C H C H C H C O O H C C H N N Fe(II) H C N N C H C H C H C H
The Heme Group - - O O C C H C H C H C H C O O 2 2 2 2 H C C H 3 3 N N Fe(II) Pyrrole ring H C N 2 N C H C H 3 C H C H C H 3 2

N of His F8 binds to 5th coordination site on heme iron
Oxygen binds to 6th coordination site on heme iron

His E7 acts as a gate to favor oxygen binding over carbon monoxide.

Hemoglobin A tetrameric protein two a-chains (141 AA)
two b-chains (146 AA) four heme cofactors, one in each chain The a and b chains are homologous to myoglobin. Oxygen binds to heme in hemoglobin with same structure as in Mb but cooperatively: as one O2 is bound, it becomes easier for the next to bind.

Hemoglobin Ubiquitous O2 transport protein
A globular soluble protein, 2X2 chains (164 kDa) a and b chains 44% identical All helical secondary structure (like myoglobin) abab quaternary structure a-subunit 141 residues b-subunit 146 residues Extensive contacts between subunits Mix of hydrophobic, H-bond, and ionic interactions a1b1 (a2b2)- 35 residues, a1b2 (a2b1)- 19 residues

Each chain is in ribbon form.
The heme groups are in space filling form

Oxygen Binding Curves Hemoglobin and myoglobin respond differently to increase in O2 concentration. Myoglobin shows normal saturation behavior while hemoglobin shows cooperative behavior. Each oxygen added to a heme of Hb makes addition of the next one easier. The myoglobin curve is hyperbolic. The hemoglobin curve is sigmoidal.

Hemoglobin O2 Binding Curve
Binding curve is sigmoidal Artery: high pO2, loading of protein Vein: lower pO2, unloading from protein P50(hemoglobin) = 26 torr, adjusts as needed!! *Drastic change in pO2 over physiological range*

Oxygen Binding Curves-2

Hemoglobin Equilibrium
a b b O2 a H+,CO2,BPG R (high affinity) T (low affinity)

A Quaternary Structure Change
One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding This large change is caused by movement of Fe by only nm when oxygen binds

Oxygen binding by hemoglobin

Allosteric Effectors The R or T state can be stabilized by the binding of ligands other than O2. H+. Lower pH favors the T state which causes Hb to release bound O2. This is known as the Bohr Effect. CO2. Release of CO2 lowers pH via conversion to HCO3-: CO2 + H2O  HCO3- + H+. Reinforces Bohr Effect Bisphosphoglycerate (BPG). Regulation of activity via binding more strongly to T state, helps to release O2. Increase in levels of BPG helps adaptation to high altitude- faster than making more hemoglobin. Also important in hypoxia diseases (e.g. anemia)

Competition between oxygen and H+
The Bohr Effect Competition between oxygen and H+ Discovered by Christian Bohr Binding of protons diminishes oxygen binding Binding of oxygen diminishes proton binding Important physiological significance-O2 saturation of Hb responds to pH

The Bohr Effect

Carbon dioxide diminishes oxygen binding
Bohr Effect II Carbon dioxide diminishes oxygen binding CO2 produced in metabolically active tissue (requires oxygen) Hydration of CO2 in tissues and extremities leads to proton production CO2 + H2O  HCO H+ These protons are taken up by Hb forcing more oxygen to dissociate The reverse occurs in the lungs

Carbon Monoxide Poisoning
Heme Fe(II) binds many other small molecules with structures similar to O2 including: CO, NO, H2S O2 is actually binds to these other molecules, particularly CO. • When exposed to CO, even at low concentrations, O2 transport proteins will be filled with CO  limiting their vital O2 capacity.

2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin The sigmoid binding curve is only observed in the presence of 2,3-BPG Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector

2,3-bisphosphoglycerate (2,3-BPG) is a negative allosteric effector of O2 binding to Hb - binds tighter to deoxyHb 2,3-BPG

Side view of Hb tetramer
Heme in hemoglobin a b b a Heme prosthetic group Side view of Hb tetramer

Binding of oxygen to heme iron
Ferrous is reduced and +2 charge Ferric is oxidized and +3 charge

Effect of oxygen on heme iron

Cooperativity Oxygen binding to one subunit of Hb, increases the affinity of the other subunits for additional oxygens. In other words, the first one is the hardest, the rest are easy. Example: square of postage stamps. Book of four stamps. To pull first stamp, you have to break two edges. To pull second stamp, you have to break only one edge. To pull third stamp, you have to break only one edge. To pull fourth stamp, you don’t have to break any edges.

Sigmoid shape indicates positive cooperativity
Mb Hb Sigmoid shape indicates positive cooperativity

Bohr Effect O2 level in arterial blood O2 level in venous blood 7.4
7.0 O2 level in arterial blood O2 level in venous blood

Hb structural families
Alpha family - a1, a2 - found in adult hemoglobins HbA1, HbA2. z - found in embryonic hemoglobins Hb Gower 1 and Hb Portland. Beta family - b - found in adult hemoglobin HbA1. d - found in adult hemoglobin HbA2. g - found in fetal hemoglobin HbF. e - found in embryonic hemoglobin Hb Gower 1 and Hb Gower 2

CO2 effect

Effect of BPG BPG is responsible for cooperativity.
High altitude increases BPG, pushing curve further to right

Effect of BPG a b Side view (T) a b BPG b a Side view (R)

Effect of BPG His+ Lys+ - BPG His+ Lys+

Hemoglobin Equilibrium
a b b O2 a H+,CO2,BPG R (high affinity) T (low affinity)

Hemoglobins in normal adults
α α γ β α δ γ β α α δ α HbA HbF HbA2 98% ~1% <3.5%

Globin gene clusters

Hb structural families
Alpha family - a1, a2 - found in adult hemoglobins HbA1, HbA2. z - found in embryonic hemoglobins Hb Gower 1 and Hb Portland. q - (theta) newly discovered embryonic form. Beta family - b - found in adult hemoglobin HbA1. d - found in adult hemoglobin HbA2. g - found in fetal hemoglobin HbF. e - found in embryonic hemoglobin Hb Gower 1 and Hb Gower 2

FETAL AND NEONATAL ERYTHROPOIESIS
TABLE 1. Globin-chain development and composition Developmental stage Hemoglobin type Globin-chain composition Embryo Embryo to fetus Fetus to adult Adult Gower 1 Gower 2 Portland Fetal A A2 Zeta2 , epsilon2a Alpha2, epsilon2 Zeta2, gamma2 Alpha2, gamma2 Alpha2, beta2 Alpha2, delta2 Alpha2, gamma2b a This tetramer may be an epsilon tetrad. b Fetal hemoglobin produced by adults has a different amino acid heterogeneity of the gamma chain at the 136 position than fetal hemoglobin

Inherited Hemoglobin disorder
Definition: An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis

The Thalassemias (quantitative)
Syndromes in which the rate of synthesis of a globin chain is reduced beta thalassemia - reduced beta chain synthesis alpha thalassemia – reduced alpha chain synthesis

Alpha Thalassemias Rare, since a gene is duplicated (four genes per diploid). Usually more severe than beta thalassemia because there is no substitute for a gene in adults. Almost all a thalassemias are deletions In a thalassemia major (a0a/a0a0) - occurrence of HbH (b4) and Hb Bart’s (g4). BPG is ineffective in HbH & Hb Bart’s.

Beta thalassemia Impaired production of beta chain
beta thalassemia minor – heterozygous (or trait) beta thalassemia major - homozygous

Beta thalassemia - heterozygous (minor or trait)
Target cell Oval cell

Beta thalassemia major

Beta Thalassemias More common, since b gene is present in only one copy per chromosome. Less severe than a thalassemia, since d chain can effectively substitute in adults. The g chain can also persist into adulthood (HPFH). In bd thal major (bd0/bd0) excess a chains do not form soluble homotetramers.

Beta thalassemia major
No beta chain produced (no HbA) Severe microcytic anemia occurs gradually in the first year of life Marrow expansion Iron overload Growth failure and death

Alpha thalassemia / Normal /- Mild microcytosis /- - -/- -
Hemoglobin H disease - -/- - Hemoglobin Barts – Hydrops Fetalis

Structural hemoglobinopathy (qualitative)
Amino acid substitution in the globin chain e.g. sickle hemoglobin (HbS)

Sickle cell hemoglobin

Red Blood Cells from Sickle Cell Anemia
Deoxygenation of SS erythrocytes leads to intracellular hemoglobin polymerization, loss of deformability and changes in cell morphology. OXY-STATE DEOXY-STATE

Sickle Cell Anemia – blood film
Sickle Cells Erythroblasts Howell-Jolly Body

Fibres of Sickle Hemoglobin

Fibres of Sickle Hemoglobin – cross section

Hemoglobin S Valine is exposed in deoxy-Hemoglobin

Polymerization of HbS

Amino Acids, Peptides, and Proteins

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