PROTEINS

Amino acids, peptides, and proteins
Pharmaceutical Chemistry 126 Lecture 3

Introduction
Peptides

and proteins polymers of amino acids linked together by amide bonds Repeating units are called amino acid residues

Introduction
dipeptide

- two amino acid

residues oligopeptide - contains three to 10 residues polypeptide proteins - naturally occurring polypeptides made up of 40 to 4000 amino acid residues

Introduction
Functions Keratin

– component of hair, horns, hooves, feathers, fur Collagen – component of bones, muscles, and tendons Snake venoms and plant toxins – protect their owners from other species

Introduction
Blood-clotting

proteins – protect the vascular system when it is injured Antibodies and protein antibiotics – protect us from disease Enzymes – catalyze chemical reactions that occur in living systems Hormones Physiological functions – transport and storage of oxygen in the body and contraction of muscles

Outline

Amino acids 1.Classification and nomenclature 2.Configuration 3.Acid-base properties 4.Separation techniques 5.

Amino acids
αamino -

acids
Other functional group R O

Amino Group

H3N+

Cα H

C O-

Carboxyl group

Standard Amino Acids Essential

Non-Essential

Arginine Arginine  Histidine Isoleucine  Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

Arg His Ile Leu Lys Met Phe Thr Trp Val

R H I L K M F T W V

Alanine Asparagine Aspartate Cysteine Glutamate Cysteine Glutamine Glycine Proline Serine Tyrosine

Ala Asn Asp Cys Glu Gln Gly Pro Ser Tyr

A N D C E Q G P S Y

Tyrosine

Classification

Classification Based on R-Groups 1.Nonpolar (hydrophobic) 2.Polar
a.Uncharged b.Charged
1)negatively charged (acidic) 2)positively charged (basic)

Hydrophobic Amino Acids
O
-

O CH3
+ NH3

-

CH3 CH3
+ NH3

O O

-

O CH3 NH3 CH3
+

-

CH3
+

O

O

O NH3
Valine(V, Val)

CH3

Alanine(A, Ala)

Isoleucine (I, Ile)

Leucine (L, Leu)

O O

-

O S
+ NH3

-

O O
+ NH3

-

CH3

O

NH3

+

H N
N H

O OH

Methionine(M, Met)

Phenylalanine(F,Phe)

Tryptophan(W, Trp)

Proline(P, Pro)

Polar Uncharged
O
-

O OH
+ NH3

-

CH3 OH
+ NH3

O O

-

O

O

NH3

+

OH

Serine (S, Ser)

Threonine (T, Thr)

Tyrosine (Y, Tyr)

O O

-

O NH2
+ NH3

-

NH2 O
+ NH3

O O

-

O SH
+ NH3

-

O

O

NH3 H

+

O

Asapragine(N, Asp)

Glutamine(Q, Gln)

Cysteine(C, Cys)

Glycine(G, G

Positively Charged
O O
+ NH3 -

NH2 NH NH2
+

O O

-

NH3 NH3
+

+

Arginine (R, Arg)

Lysine (K, Lys)

O O

-

H N NH3
+

N H

+

Histidine (His, H)

Negatively Charged

O O

-

O NH3
+

-

O O

-

O

-

O NH3
+

O

Aspartic Acid (D, Asp)

Glutamic Acid (E, Glu)

Configuration

Optical activity
All

AAs are optically active EXCEPT glycine All AAs found in nature have Lconfiguration D L-cysteine alanine AllL-L-amino acids EXCEPT -alanine O has S configuration O

H2N OH H CH3
H

OH NH2

CH3

S Prioritie NH2 > s COOH> Alkyl> H

R

Optical activity
L-cysteine

O OH H2N H SH

R Prioritie s NH2 > COOH> Alkyl> H

Acid-base properties

Isoelectric point

Isoelectric point

Separation techniques
1.Electrophoresis

Separation techniques
2.Paper and thin-layer chromatography

S e p a ra ti n o f A m i o A ci s o n d
E l ctro p h o re si e s
a. +

anode
pI = 2.98

Chromatography
Least polar

pI = 6.02

O OO

- O

O O

CH3 O NH + NH3 O

pI = 10.76
NH2 NH2
+

O O OOO
O CH3 O CH
3

- -

Most polar
+

+ + NH3 3 NH

O O O + H

O OH NH2 R

H2O

cath ode

NH O NH3 3 CH3

+ + NH3

O N O

O

stationary phase: more polar mobile phase: less polar

O

Separation techniques
3.Ion-exchange chromatography 4. 5. 6. 7. 8. 9. Cation-exchange resin

Separation techniques

Separation techniques

Synthesis of amino acids
Hell-Volhard-Zellinski 

reaction

Synthesis of amino acids
Show

how you could prepare the following α -amino acids from the appropriate carboxylic acids: 1.Phenylalanine 2.Valine

Synthesis of amino acids
α-amino

acids can be prepared by treating an aldehyde with ammonia and hydrogen cyanide, followed by acid-catalyzed hydrolysis 1.Give the structures of the two intermediates formed in this reaction. 2.What amino acid is formed when the aldehyde that is used is 3methylbutanal? 3.What aldehyde would be needed to prepare valine?

4.

Synthesis of amino acids
Amidomalonate

synthesis

Synthesis of amino acids
What

alkyl halides would you use to prepare the following αamino a ci s b y th e a m i o m a l n a te d d o m e th o d ? 1 . Le u ci e n 2 .H i d i e sti n 3 . Tryp to p h a n 4 . M e th i n i e o n

Synthesis of amino acids
Reductive

a ci s d

amination of αketo -

Resolution of racemic mixtures
Kinetic

resolution

Outline

Peptides 1.Peptide and disulfide bonds 2.Peptide synthesis 3.

Peptide bonds

Peptide bonds

Peptide bonds

Peptide bonds
1.Name the following peptides: HO O 2. NH O O O NH H N 3. O NH NH NH O 4. OH CH CH 5. H C HO 6. 7.Draw the following peptides:  P-H-A-R-M  C-H-E-M
2 2 3 3 3

NH

Peptide bonds

Peptide bonds

Disulfide bonds

Disulfide bonds

Disulfide bonds

Peptide bond synthesis

Outline

Proteins 1.Protein structure 2.Protein denaturation

Protein structure
Levels

of protein structure: 1.Primary structure
◦ Sequence of amino acids in the chain and location of all disulfide bridges

2.Secondary structure
◦ Describes regular conformation assumed by segments of the protein’s backbone ◦ Describes how local regions of the backbone fold

Protein structure
Levels

of protein structure: 3.Tertiary structure
◦ Describes 3D structure of the entire polypeptide

5.Quaternary structure
◦ Applicable if a protein has more than one polypeptide chain ◦ Describes the way the individual protein chains are arranged with respect to each other

Protein structure
Fibrous Globular

Determination of primary structure
1.Reduce any disulfide bridge present
◦ 2-mercaptoethanol ◦ Iodoacetic acid

Determination of primary structure
2.Determine the number and kinds of amino acids present in the peptide or protein 3. 4. Hydrolyzes all amide bonds including those of asparagine and glutamine

Determination of primary structure
Mixture

of amino acids is then passed through an amino acid analyzer # of Asp residues = # of Asp + Asn residues # of Glu residues = # of Glu + Gln residues Acid hydrolysis destroys Trp residues; Trp content can be determined by basic hydrolysis

Determination of primary structure
3.Determine N-terminal residue Phenyl isothiocyanate (PITC) or Edman’s reagent N-terminus + PITC = thiazolinone derivative Thiazolinone derivative is cleaved from protein, extracted into organic solvent, rearranges to a phenylthiohydantoin

Determination of primary structure
The

particular PTH formed can be identified by chromatography using known standards Sequenator – automated instrument which allows 50 successive Edman degradations to be carried out

Determination of primary structure
4.Determine C-terminal residue
◦ Carboxypeptidase A
 Cleaves off C-terminal amino acid as long as it is not Arg or Lys

◦ Carboxypeptidase B
Cleaves off C-terminal amino acid only if it is Arg or Lys

Determination of primary structure
5.Hydrolyze protein with dilute acid
◦ Partial hydrolysis ◦ Fragments are separated, amino acid composition of each is determined ◦ N-terminal and C-terminal amino acids of each fragment is identified ◦ Entire sequence   u    t   s          r     ts   vr

Determination of primary structure
A nonapeptide undergoes partial hydrolysis to give peptides whose amino acid compositions are shown. Reaction of the intact nonapeptide with Edman’s reagent releases PTH-Leu. What is the sequence of the

a.Pro, Ser b.Gly, Glu c.Met, Ala, Leu d.Gly, Ala e.Glu, Ser, Val, Pro f. Glu, Pro, Gly g.Met, Leu h.His, Val

Determination of primary structure
A decapeptide undergoes partial hydrolysis to give peptides whose amino acid compositions are shown. Reaction of the intact decapeptide with Edman’s reagent releases PTH-Gly. What is the sequence of the

a.Ala, Trp b.Val, Pro, Asp c.Pro, Val d.Ala, Glu e.Trp, Ala, Arg f. Arg, Gly g.Glu, Ala, Leu h.Met, Pro, Leu, Glu

Determination of primary structure

Determination of primary structure
Trypsin 

Determination of primary structure
Trypsin   Chymotrypsin

– cleaves at C-side of only arginine or lysine residues

– cleaves at C-side of amino acids that contain aromatic six-membered rings (Phe, Tyr, Trp)

 

Determination of primary structure
Elastase   None

– cleaves at C-side of small amino acids (Gly, Ala)

of the mentioned will catalyze the hydrolysis of an amide bond if proline is at the hydrolysis site

Determination of primary structure
Cyanogen

bromide

◦ Causes hydrolysis of amide bond on the C-side of a methionine residue ◦ Will still cleave the peptide bond if proline is at the cleavage site

Determination of primary structure
6.Determinine location of any disulfide bonds Hydrolysis of protein with intact disulfide bonds Determine amino acids in Cyscontaining fragments

Determination of primary structure

Determination of primary structure

Secondary structure of proteins
Describes

conformation of segments of the backbone chain of a peptide or protein αhelix β -pleated sheet

 α-helix

Secondary structure: αhelix

 α-helix

Secondary structure: αhelix

Secondary structure of proteins
α -helix

◦ Backbone of the polypeptide coils around the long axis of the protein molecule ◦ Stabilized by H bonds ◦ Each hydrogen attached to an amide nitrogen is H-bonded to carbonyl oxygen of an amino acid four residues away ◦ R groups protrude outward from the helix, minimizing steric hindrance ◦

Secondary structure of proteins
α-helix

◦ Right-handed helix
 Rotates in a clockwise direction as it spirals down Because of L-amino acids

◦ Each turn of the helix contains 3.6 amino acid residues ◦ Pro residue forces a bend in the helix
Bond between Pro nitrogen and αcarbon cannot rotate

 α-helix

Secondary structure: βpleated sheet

 α-helix

Secondary structure: βpleated sheet

 α-helix

Secondary structure: βpleated sheet

 α-helix

Secondary structure: βpleated sheet

Secondary structure of proteins
βpleated -

sheet

◦ polypeptide backbone is extended in a zigzag structure resembling a series of pleats ◦ almost fully extended ◦ H-bonding occurs between neighboring peptide chains ◦ adjacent hydrogen-bonded peptide chains can run in the same direction (parallel) or in opposite (antiparallel) directions ◦ R groups – close to each other;

Tertiar y structu re of protein s

Tertiar y structu re of protein s

Tertiary structure of proteins
Overall

3D shape of a protein Forces that stabilize structure: 1.Covalent bonds (disulfide links) 2.Ionic bonds 3.Hydrogen bonds 4.Van der Waals or hydrophobic interactions

Tertiary structure of proteins
Covalent

bond

Tertiary structure of proteins
Ionic

bond

Tertiary structure of proteins
Hydrogen

bond

Tertiary structure of proteins
Van

der Waals interaction

Tertiar y structu re of protein s

Tertiary structure of proteins
Determination

of structure

◦ X-ray crystallography ◦ Magnetic Resonance Imaging ◦ 2-D NMR Spectroscopy ◦

Quaternary structure of proteins
Oligomers

– proteins that have more than one peptide chain Subunits – individual chains Monomer – protein with a single subunit Dimer – two subunits Trimer – three subunits Tetramer – four subunits  ex. Hemoglobin

Quaternary structure of proteins
Subunits

are held together by hydrophobic interactions, Hbonding, and ionic attractions

    
Quaternary

structure describes the way subunits are arranged in space

Quaternary structure of proteins
Possible 

arrangements of the six subunits of a hexamer:

Protein denaturation
Destruction

of tertiary structure Bonds responsible for maintaining the 3D shape of the protein are broken

Random coil

Protein denaturation
 Change

in pH: alters charges on side chains; disrupts electrostatic attractions and H-bonds  Urea and guanidine HCl: forms H-bonds to protein groups that are stronger than the H-bonds formed between the groups  Detergents (sodium dodecyl sulfate): associates with nonpolar groups of the protein; interferes with normal hydrophobic interactions  Organic solvents: disrupts hydrophobic interactions

Applications to the pharmaceutical sciences

Proteins as Drug Targets Enzymes Receptors Carrier Proteins Structural Proteins Proteins or Peptides as Drugs Polypeptide hormones Immunoglobulins and antigens Polypeptide antibiotics Natural toxins


End of lecture 

Sign up to vote on this title
UsefulNot useful