Proteins are polymers composed of amino acids joined by peptide bonds. There are 20 genetically encoded amino acids that vary in properties like polarity, charge, and solubility. Protein structure is organized in four levels - primary, secondary, tertiary, and quaternary. The primary structure is the amino acid sequence, which determines the higher order structures that influence a protein's function, such as enzyme catalysis or oxygen transport. Sanger's method uses 1-fluoro-2,4-dinitrobenzene to label the N-terminal amino acid and determine protein sequence.

1. Chemistry of protein
1
Proteins are polymers of amino acids, with
each amino acid residue joined to its neighbor
by a specific type of covalent bond.

4. Chemical composition of proteins.
Proteins are composed of:
 Carbon 50-55%
 Oxygen 21-24%
 Nitrogen 15-18%
 Hydrogen 6.5-7.3%
 Sulphur 0.0-2.4%
Some proteins may contain phosphorus and/or metals.
Complete hydrolysis results in amino acids (total of 20).
Proteins are polymers composed of amino acids.

11. • At physiological PH (7.4), -COOH group is dissociated forming a negatively charged
carboxylate ion (COO-) and amino group is protonated forming positively charged ion
(NH3
+) forming Zwitter ion
Classification of amino acids
I- Chemical classification: According to number of COOH and NH2 groups i.e. according
to net charge on amino acid.
A- Monobasic, monocarboxylic amino acids i.e. neutral or uncharged:
R

12. Subclassification of neutral amino acids:
1- Glycine R= H
2- Alanine R= CH3
3- Branched chain amino acids:
a - Valine
b- Leucine
c- Isoleucine
4- Neutral Sulfur containing amino acids:
e.g. Cysteine and Methionine. What is cystin?
5- Neutral, hydroxy amino acids:
e.g. Serine and Threonine

13. 6- Neutral aromatic amino acids:
a- Phenyl alanine
b- Tyrosine
c- Tryptophan
7- Neutral heterocyclic amino acids:
a- Tryptophan: contains indole ring
b- Proline

14. B- Basic amino acids: Contain two or more NH2 groups or nitrogen atoms that act as base i.e. can bind
proton.
At physiological pH, basic amino acids will be positively charged.
e.g.
a- Lysine
b- Arginine: contains guanido group
c- Histidine: is an example on basic heterocyclic amino acids

15. C- Acidic Amino acids: at physiological pH will carry negative charge.
e.g. Aspartic acid (aspartate) and Glutamic acid (glutamate).
Aspargine and Glutamine: They are amide forms of aspartate and glutamate in which
side chain COOH groups are amidated.
They are classified as neutral amino acids.

16. 16
Classification of Amino Acids. AA’s are classified
according to the location of the amino group.
There are 20 genetically encoded-amino acids found in peptides
and proteins
19 are primary amines, 1 (proline) is a secondary amine
19 are “chiral”, 1 (glycine) is achiral; the natural configuration of
the -carbon is L.

17. Properties of α – amino carboxylic acids
Stereochemistry:
For all amino acids except for glycine, α-carbon atom is chiral:
bonded to four different groups - a carboxyl-, an amino-, a R- groups
and a hydrogen atom.
Two stereoisomers: enantiomers
Mirror images: L- and D- forms
Carbons are lined up vertically, with
the chiral atom in the center.
When α-amino group is on the left
site of the vertical line, the AA is L-
form.
When α-amino group is on the right
site of the vertical line, the AA is D-
form.

18. Two enatiomers possible for most amino
acids
L-form found almost exclusively in naturally occurring proteins

19. 20 Amino Acids
Nonpolar,
hydrophobic
Polar,
uncharged
Polar,
charged

20. Some proteins may contain non-standard amino acids.
5-hydroxylysine

21. III- Nutritional classification:
1- Essential amino acids: These amino acids can’t be formed in the body and so, it is
essential to be taken in diet. Their deficiency affects growth, health and protein
synthesis.
2- Semiessential amino acids: These are formed in the body but not in sufficient
amount for body requirements especially in children.
Summary of essential and semiessential amino acids:
V= valine i= isoleucine l= lysine l= leucine
A = arginine* H= histidine* M= methionine
T= tryptophan Th= threonine P= phenyl alanine
*= arginine and histidine are semiessential
3- Non essential amino acids: These are the rest of amino acids that are formed in the
body in amount enough for adults and children. They are the remaining 10 amino
acids.

22. Essential Amino Acids

23. IV- Metabolic classification: according to metabolic or degradation products of amino
acids they may be:
1- Ketogenic amino acids: which give ketone bodies . Lysine and Leucine are the only
pure ketogenic amino acids.
2- Mixed ketogenic and glucogenic amino acids: which give both ketonbodies and
glucose.These are: isoleucine, phenyl alanine, tyrosine and tryptophan.
3- Glucogenic amino acids: Which give glucose. They include the rest of amino acids.
These amino acids by catabolism yields products that enter in glycogen and glucose
formation.

24. Amphoteric properties of amino acids: that is they have both basic and acidic groups
and so can act as base or acid.
Neutral amino acids (monobasic, monocarboxylic) exist in aqueous solution as “
Zwitter ion” i.e. contain both positive and negative charge. Zwitter ion is electrically
neutral and can’t migrate into electric field.
Chemical properties of amino acids:
1- Reactions due to COOH group:
-Salt formation with alkalis, ester formation with alcohols, amide formation with
amines and decarboxylation
2- Reactions due to NH2 group: deamination and reaction with ninhydrin reagent.
-Ninhydrin reagent reacts with amino group of amino acid yielding blue colored
product. The intensity of blue color indicates quantity of amino acids present.

25. Ninhydrine can react with imino acids as proline and hydroxy proline but gives
yellow color.
3- Reactions due to side chain (R):
1- Millon reaction: for tyrosine gives red colored mass
2- Rosenheim reaction: for trptophan and gives violet ring.
3- Pauly reaction: for imidazole ring of histidine: gives yellow to reddish product
4- Sakagushi test: for guanido group of arginine andgives red color.
5- Lead sulfide test (sulfur test): for sulfur containing amino acids as cysteine give
brown color.

26. PROTEIN FUNCTION

27. A protein’s function depends on its specific conformation
• A functional proteins consists of one or more polypeptides that have been
precisely twisted, folded, and coiled into a unique shape.
• It is the order of amino acids that determines what the three-dimensional
conformation will be.

28. • In almost every case, the function depends on its ability to recognize and
bind to some other molecule.
• For example, antibodies bind to particular foreign substances that fit their
binding sites.
• Enzyme recognize and bind to specific substrates, facilitating a chemical
reaction.
• Neurotransmitters pass signals from one cell to another by binding to receptor
sites on proteins in the membrane of the receiving cell.

29. Levels of Protein Structure
1. Primary structure
2. Secondary structure
3. Tertiary structure
 are used to organize the folding within a single polypeptide.
4. Quarternary structure arises when two or more polypeptides join
to form a protein.

30. • The primary structure of a protein is
its unique sequence of amino acids.
• Lysozyme, an enzyme that attacks
bacteria, consists on a polypeptide
chain of 129 amino acids.
• The precise primary structure of a
protein is determined by inherited
genetic information.

31. • Even a slight change in primary structure can affect a protein’s
conformation and ability to function.
• In individuals with sickle cell disease, abnormal hemoglobins,
oxygen-carrying proteins, develop because of a single amino acid
substitution.
• These abnormal hemoglobins crystallize, deforming the red blood cells and
leading to clogs in tiny blood vessels.

32. • The secondary structure of a
protein results from hydrogen
bonds at regular intervals along
the polypeptide backbone.
• Typical shapes that develop from
secondary structure are coils (an
alpha helix) or folds (beta
pleatedsheets).

33. • Tertiary structure is determined by
a variety of interactions among R
groups and between R groups and the
polypeptide backbone.
• These interactions include hydrogen
bonds among polar and/or charged
areas, ionic bonds between charged R
groups, and hydrophobic interactions
and
van der Waals interactions among
hydrophobic R groups.

34. • Quarternary structure results from the
aggregation of two or more polypeptide
subunits.
• Collagen is a fibrous protein of three polypeptides
that are supercoiled like a rope.
• This provides the structural strength for their role
in connective tissue.
• Hemoglobin is a
globular protein
with two copies
of two kinds
of polypeptides.

35. Amino acid solubility
 Nonpolar, hydrophobic: tend to cluster together within proteins,
stabilize protein structure by the means of hydrophobic interactions.
They are not soluble in water.
 Polar with uncharged and charged R-groups are hydrophilic and
soluble in water because they contain functional groups that form
hydrogen bonds with water.

36. 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.

37. 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.
FO2N
NO2

38. Sanger's Method
• 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-
terminal amino acid.
FO2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
H2NCHC
O OOO
CH(CH3)2
–
+

39. Sanger's Method
• 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal
amino acid.
FO2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
H2NCHC
O OOO
CH(CH3)2
–
+
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–

40. 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.
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–

41. 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.
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–
H3O+
O
O2N
NO2
NHCHCOH
CH(CH3)2

42. 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.
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–
H3O+
H3NCHCO–
CH3
+
H3NCH2CO–
O OO
O2N
NO2
NHCHCOH
CH(CH3)2
+
O
H3NCHCO–
CH2C6H5
+
+ +
+

43. Denaturation
• Denaturation is a phenomenon that involves transformation of
a well-defined, folded structure of a protein, formed under
physiological conditions, to an unfolded state under non-
physiological conditions.
• Occurs suddenly and completely over a narrow range of conditions
• Slowly reversible (if at all)

44. • A protein’s conformation can change in response to the physical and
chemical conditions.
• Changes in pH, salt concentration, temperature, or other factors can
unravel or denature a protein.
• These forces disrupt the hydrogen bonds, ionic bonds, and disulfide bridges that
maintain the protein’s shape.
• Some proteins can return to their functional shape after denaturation,
but others cannot, especially in the crowded environment of the cell.
• Usually denaturation is permanent

46. • In spite of the knowledge of the three-dimensional shapes of over
10,000 proteins, it is still difficult to predict the conformation of a
protein from its primary structure alone.
• Most proteins appear to undergo several intermediate stages before reaching
their “mature” configuration.

47. Types of Denaturation
• Temperature
• Organic solvents
• Surface
• pH
• Shear

48. Thermal Denaturation
• Trypsinogen 55°C
• Pepsinogen 60°C
• Lysozyme 72°C
• Myoglobin 79°C
• Soy Glycinin 92°C
• Oat globulin 108°C
Table 11
Affected by pH, water,
solutes

49. Denaturation may not require complete unfolding of proteins. It might be still a
folded structure but in random conformation.
Denaturation is cooperative, I.e. changes in one part of protein acelerate the unfolding
of the other part.
Some proteins are resistant to denaturation by heat (Proteins of hot spring
bacteria stable at 100 oC). The primary structure of these proteins are not very
different from those from normal bacterium. It remains a biochemical puzzle to
explain the stability these proteins.
Heat: destabilizes H-bonding
Detergents, Urea, organic solvents: destabilize hydrophobic interactions
Extreme pH conditions: cause ionization of side chains resulting in electrostatic
repulsion and collapse of structure.

50. Christian Anfinsen, 1950: Denaturation of ribonuclease by urea and reducing agent led
to complete loss of activity.
Removing the urea and reducing agent from this mixture resulted in complete
renaturation of this enzyme.

51. Behavior of Denatured Protein
Hydrophobic core
Hydrophilic surface
NATIVE
AGGREGATED
or other ingredient interactions
DENATURED
Unfolding forces some
hydrophobic AA to surface
Fast under non-physiological conditions
Slow under physiological conditions

52. Consequences of Denaturation
• Loss of enzymatic activity (death)
• Destruction of toxins
• Improved digestibility
• Loss of solubility
• Changes in texture

53. Renaturation of Ribonuclease

54. A computer simulated pathway of folding of villin protein
(36AA long polypeptide)

55. Diseases caused by the defect in protein folding:
Cystic fibrosis: Defect in the folding of cystic fibrosis Tran membrane conductance
regulator protein.
Diseases caused by misfolding of Prion proteins:
Kuru Disease
Creutzfedlt-Jakob Disease
Scrapie Disease in sheep
Mad cow disease
Misfolded prion protein act as infectious agents.
They act as chaperons which can multiply by binding to normal PrP and folding it to
dangerous form similar to itself.
Mechanisms of the functions of normal prions and the dangerous ones are still not clear.