Alissa Laurel N.

Calderon, MD, FPAFP, MMHA

Proteins
“Proteios” – primary, holding first place, of first
importance.
 Most abundant and functionally diverse
molecules in living systems
 are complex, organic nitrogenous
substances with very high molecular
weights, found in all plant and animal cells
 consisting largely or entirely of alpha-amino
acids united in peptide linkage.
 AMINO ACID
• building blocks of
proteins
 PROTEINS
• more than 50
amino acids
 OLIGOPEPTIDES
• 2-10 AA
 POLYPEPTIDES
• >10 AA residues
Based on compositions, physical and chemical
properties of the proteins
SIMPLE PROTEINS
• Albumin – soluble in water and dilute aqueous salt
solutions, heat coagulable
• Globulin – insoluble in water, soluble in aqueous
salt solution, heat coagulable
• Glutelin – soluble in dilute acid and alkalies, heat
coagulable
• Prolamine – alcohol soluble protein
• Albuminoid or scleroprotein – least soluble
• Histone – basic protein; soluble in water, dilute acid
and alkali, found in combination with DNA
Based on compositions, physical and
chemical properties of the proteins
 CONJUCATED PROTEINs
• Nucleoprotein
• Glycoprotein
• Phosphoprotein
• Chromoprotein
• Lipoproteins
• Metalloproteins
BASED on shape and certain physical
characteristics of the protein
FIBROUS PROTEINS
• They are tough, insoluble in water
• They are arranged around a single axis to form a
fiber
• They are involved in structural functions
• COLLAGEN – most abundant, 25% protein of the
body
• KERATIN- hair, scales,horns, wool, nails and
feathers
 GLOBULAR
PROTEINS
• Involved in mobile
and dynamic functions
• Enzymes, hemoglobin,
plasma proteins
BASED on Biologic Functions
 ENZYMES
 STORAGE
 REGULATORY
 STRUCTURAL
 PROTECTIVE
 TRANSPORT
 CONTRACTILE/MOTILE
 To understand protein function, we must
first understand the nature of amino
acids.
 Amino acids are essentially α-amino
acids:
alpha carbon (IUPAC #2 position)

H2N – C – COOH
|
R
 When R is not H, the alpha carbon
is asymetric, giving rise to isomers.
Amino Acids
 building block of proteins
 has a central carbon, called the alpha carbon,
to which four different groups are attached:

1. a basic amino group (-NH2)

2. an acidic carboxyl group (-COOH)
3. a hydrogen atom (-H)
4. a distinctive side chain (-R)

 -carbon is asymmetric (except Glycine)

 when R is not H, the alpha carbon is
asymetric, giving rise to isomers
1. Building blocks of proteins
2. Precursor of various substances
a. heme, purine, creatine, glutathione,
hippuric acid
b. epinephrine, melanin
c. niacin, serotonin, melatonin, indole &
skatole,melatonin
d. Histamine
e. GABA
3. Source of energy
4. Special amino acids as components of
certain types of proteins
a. collagen
b. prothrombin
c. Desmosine (derivative of lysine)
- elastin
d. myosin
 Functions of A. A.
5. Phosphorylation and dephosphorylation
of Ser, Thr and Tyr play major role in
activation and inactivation of enzymes.
6. Some amino acids or their derivatives
act as chemical messengers
7. Several amino acids act as metabolic
intermediates [e.g. Arg, citrulline, ornithine
– urea cycle]
Classification of A. A. Based on
Polarity

I. Amino acids with nonpolar or hydrophobic R

groups
- - play an impt. role in maintaining the
conformation or 3-dimensional structure of
proteins

A. Amino acids with Aliphatic side chains

B. Amino acids with Aromatic side chains
Classification of A. A.
[Non-polar…]
A. Amino acids with aliphatic side chains
1. Glycine
2. Alanine

Branched-chain amino acids

1. Valine
2. Leucine
3. Isoleucine
4. Methionine
5. Proline
Classification of A. A.

Alanine Glycine

 Glycine is the simplest amino acid.

Classification of A. A.

Branched-chain amino acid

Leucine Valine Isoleucine

 Methionine Proline

 S-adenosylmethionine (SAM) is the methyl group

donor in methylation reactions.
 Proline has a secondary amino group referred to as
-imino acid; nitrogen is bonded to the α-carbon
and the side-chain group
- also known as “helix-breaker”
Classification of A. A
[Non-polar…]
B. Amino acid with aromatic side chains
1. Phenylalanine
2. Tryptophan

Phenylalanine Tryptophan
Classification of A. A.
II. Amino acids with uncharged polar R groups
- have functional groups capable of hydrogen
bonding with water
- Important factor in protein structure

A. Hydroxyl-containing amino acids

B. Amide derivatives of Glu and Asp
C. Sulfhydryl-containing amino acids
Classification of A. A.
[Polar uncharged…]
A. Hydroxyl-containing amino acids
1. serine
2. threonine
3. tyrosine

- site of phosphorylation reaction

- impt. in regulation of enzyme activity
- site of glycosylation reaction
- impt. in formation of mucin
Classification of A. A.

Hydroxyl-containing amino acids

Serine Threonine Tyrosine

Classification of A. A.
[Polar uncharged…]
B. Amide derivatives of Glu and Asp
1. glutamine
2. asparagine

- impt. in detoxification of ammonia

- glutamine is the primary source of urinary
ammonia
Classification of A. A. [cont’n…]

Amide derivatives of Asp and Glu

Asparagine Glutamine
(Amide-containing amino acids)
 Classification of A. A.

[Polar uncharged…]

Cysteine
- contains the sulfhydryl (-SH) group
- when two cysteine residues
combine a strong disulfide bond is
formed and cystine is the resulting
product
- disulfide bond is also impt. in
maintenance of protein structure Cysteine
(Thiol group)
Classification of A. A.
III. Amino acids with positively (+) charged R groups
Basic amino acids
1. lysine
2. arginine
3. histidine

- histidine contains the imidazole group

- responsible for the buffering capacity of
hemoglobin
- arginine contains the guanido group
Classification of A. A.

Basic amino acids

Lysine Arginine Histidine

Classification of A. A.
IV. Amino acids with negatively (-) charged R groups
Acidic amino acids
1. glutamic acid
2. aspartic acid
- interactions between acidic and basic amino
acids result in the formation of ionic bond
[salt bond, electrostatic bond]
- important in maintaining protein structure
Classification of A. A.

Acidic amino acids

Aspartic acid Glutamic acid

Types of Interactions
 entered into by the different R groups of amino
acids
1. Hydrogen bonding
a. - O-H …….. O=C
b. - O-H …….. O-
c. - S-H ……... O-
2. Ionic interaction -COO- ……..NH3+
3. Hydrophobic interactions of nonpolar R grps.
4. Disulfide bond
 Physical Properties of A. A.

Optical property
 For all the standard amino acids, except glycine, the -
carbon is asymmetric, i.e., -carbon is a chiral center.
 All molecules with a chiral center are optically active –
i.e., they can rotate the plane-polarized light either to the
right [dextrorotatory] or to the left [levorotatory].
 Amino Acid Chemistry

Physical Properties of A. A. [cont’n…]

Stereoisomerism – D and L amino acids
- due also to the presence of chiral center
- basis of classification is the similarity to
glyceraldehyde standard
- D isomer if NH2 group is oriented to the right
- L isomer if NH2 group is oriented to the left
- amino acids that occur in protein are all L form
- D amino acids are found in bacterial cell wall and
some antibiotics
- 2 D-amino acids found in the human brain:
D-aspartate and D-serine
Physical Properties of A. A.

D and L amino acids

Physical Properties of A. A.
6. Ultraviolet absorption spectrum of aromatic amino
acids. UV absorption properties of proteins are
determined solely by Phe, Try and Trp.

7. Acid-base properties
- The amino and carboxylic acid groups readily ionize
pK (-COOH) = pH 2.2
pK (-NH2) = pH 9.4
- An amino acid can act as a base and an acid
[amphoteric].
 Different Forms of an A. A.
1. Unionized form H H

H2N - C - COOH H3N+ - C – COO-

2. Dipolar ion or
R R
zwitterion form [0]

[1] [2]
3. Fully protonated [pH 1]

H H
4. Fully ionized [pH 11]
+H
3N - C – COOH H2N - C – COO-
R R
[+1] [-1]

[3] [4]
Different Forms of an A. A.
Zwitterion

- isoelectric or isoionic form

- electrically neutral; zero net charge
- equal number of positive and negative
charges
- remain stationary in an electrical field
Titration of Amino Acids (Protonic equilibria)

Protonic equilibria is the step by step dissociation

of the amino acid starting from the fully
protonated or positive form up to the fully
ionized or negative form.
Uses:
1. can predict the charge of the amino acid in a
given solution with known pH
2. can devise a procedure of separating amino
acids based on their charge
Titration of Amino Acids (Protonic equilibria)
Isoelectric Point (pHI, pI, IpH)
- That pH at which an amino acid bears no net
charge and hence does not move in an electrical
field.
- That pH exactly at the midpoint between the pK
values on either side of the zwitterion species.
- General rule regarding pI:
 pI of neutral amino acids - neighborhood of 6.0;
 pI of acidic amino acids - very much below 6.0;
 pI of basic amino acids - very much above 6.0.
Protonic equilibria…
Dissociation of monoamino, monocarboxylic a. a.
[Neutral amino acid]
 Formula for pI of NAA:

pI = pK1 + pK2
2
Example: alanine

pI = 2.34 + 9.69
2
= 6.02
Note:
pK1 – dissociation
constant of -COOH
(most acidic group)
Titration curve of monoamino, pK2 – dissociation
monocarboxlic a. a. [neutral a. a.] constant of -NH2
Protonic equilibria…
Dissociation of diamino, monocarboxylic a. a.
[Basic amino acid]
 Formula for pI of BAA:

pI = pK2 + pKr
2
Example: histidine

pI = 6.0 + 9.2
2
= 7.6

Note:
pKr – dissociation
constant of the
R-group
Titration curve of diamino,
monocarboxylic a. a. [basic a. a.]
Protonic equilibria…
Dissociation of monoamino, dicarboxylic a. a.
[Acidic amino acid]
 Formula for pI of AAA:

pI = pK1 + pKr
2
Example: glutamic acid

pI = 2.19 + 4.25
2
= 3.57

Note:
pKr – dissociation
constant of the
Titration curve of monoamino, R-group
dicarboxylic a. a. [acidic a. a.]
 Peptides
Peptides – chain of amino acids

- amino acid polymers with low molecular weights,

typically consisting of less than 50 a. a.
- amino acids are joined by peptide bond
- amino acid units are called residues
 How to draw?
Peptide Bond …
Parts of the peptide chain

Ex. [Ser-gly-tyr-lys-ala-leu]
Peptide Bond …

Evidences that a peptide bond exist in

proteins:
1. Positive reaction to the Biuret test
2. Relatively few titratable amino and
carboxyl functions
3. Hydrolyzed by enzymes which are
specific for the peptide bond
4. X-ray diffraction studies proved the
existence of peptide linkages between
amino acids in hemoglobin and
myoglobin
5. Synthesis of insulin
Peptides w/ biological activities
1. Glutathione (gamma-glutamyl-L-
cysteinylglycine)
- reducing agent; due to –SH group
- protects the cell from the destructive
effects of oxidation by peroxides
- works hand in hand with glutathione
peroxidase
2. Oxytocin and vasopressin
- contain nine amino acid residues
- oxytocin stimulates uterine muscle
contraction
- vasopressin is an antidiuretic
hormone; stimulates water
reabsorption in the kidney
Cys-tyr-ile-gln-asn-cys-pro-leu-gly-NH2
3. Glucagon
- has 29 amino acid residues
- opposes the action of insulin
Peptides w/ biological activities
4. Met-enkephalin and Leu-enkephalin
- pentapeptide; opioid peptides
- relieve pain; bind to receptors in the brain
and induce analgesia
Tyr-gly-gly-phe-met
5. Atrial natriuretic factor
- has 28 amino acid residues
- stimulates the production of a dilute
urine
6. Substance P and bradykinin
- stimulate the perception of pain
7. Aspartame [L-aspartylphenylalanine
methyl ester]
- artificial sweetener
 24 H in a D
 3 W on a T
 29 D in F in a LY
 52 W in a Y
Levels of Structural Organization
(Protein Structure)

A. Primary structure
B. Secondary structure
C. Tertiary structure
D. Quaternary structure
 Protein Chemistry

Levels of Structural Organization

A. Primary Structure
- refers to quantitative amino acid composition;
sequence of a. a.; number of peptide chains
- stabilized by peptide bond
- the backbone of a protein refers to the atoms that
participate in the formation of peptide bonds

 Most abundant amino acid in proteins:

[Leu, Ala, Gly, Ser, Val and Glu]
 Rarest in proteins are: [Trp, Cys, Met and His]
 Protein Chemistry

Levels of Structural Organization

B. Secondary Structure
- due to the formation of hydrogen bonds
between peptide bonds

 Two types:
1. Coils or helices
- intrachain hydrogen bonding
2. Sheets or pleats
- interchain hydrogen bonding
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]
1. Alpha-helix: discovered by Linus Pauling in 1951

Important features:
 Stabilized by inter-residue H-bonds formed bet. the H
atom attached to a peptide N and the carbonyl O atom.
 Each peptide bond participates in H-bonding.
 -helix forms spontaneously as it is the lowest energy,
most stable conformation for a polypeptide chain.
 There are 3.6 amino acid residues per turn with a pitch
(distance bet. corresponding points per turn) of .54 nm
(5.4 A); spacing per residue is .15 nm (1.5 A).
 Amino acid R groups extend outward from the helix.
 Protein Chemistry

Features of an -Helix Structure

 Protein Chemistry

Alpha-Helix Structures
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]
Factors that destabilize the alpha-helix:

1. Presence of adjacent similarly charged a. a.

2. Presence of adjacent bulky R groups.
3. Presence of proline
- contains rigid ring that prevents the N-C
bond from rotating
- no N-H group available to form intrachain
hydrogen bonds
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]
2. Beta-pleated Sheet – 2nd most commonly occurring
protein 2o structure
Important Features:
 Formed when 2 or more almost fully extended
polypeptide chains lie side by side.
 H-bonds are interchain.
 R grps lie above or below the zigzagging planes
of the pleated sheet and are nearly perpendicular
to them.
 Amino acids with less bulky R groups are present.
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]
Forms of Beta-pleated Sheet
1. Antiparallel beta-sheet
- neighboring H-bonded polypeptide chains
run in opposite directions
- more stable
2. Parallel beta-sheets
- H-bonded chains extend in the same direction.
 Protein Chemistry

Antiparallel beta-pleated sheet Parallel beta-pleated sheets

 Protein Chemistry

Beta-Pleated Sheets
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]
Factors that destabilize the β-pleated sheets:

1. Bulky R groups.
2. R groups with like charges.
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]

Supersecondary Structures or Motifs

- Combinations of 2o structure
- Occur as part of a larger functional unit
- Have different functions in different proteins
- Have a particular function
- Have roughly 10 to 40 a. a. residues each
 Protein Chemistry

Levels of Structural Organization

[2 Structure …]

Common Supersecondary Structures

1. Helix-loop-helix
2. Coiled-coil motif
3. Beta-alpha-beta unit
4. Hairpin
5. Zinc finger
6. Leucine zipper
7. Greek key
 Protein Chemistry

Beta-alpha-beta units

Beta-meander

Alpha-alpha units

Beta-barrel

Greek key

Supersecondary structures
 Protein Chemistry

EF hand Zinc
finger

Leucine
zipper

Supersecondary structures
 Protein Chemistry

Levels of Structural Organization

C. Tertiary Structure

- 3-dimensional structure
- Protein conformation
- results from folding of a polypeptide
- indicates how 2o structural features – helices,
sheets, bends, turns and loops assemble to
form domains.
- the 3-dimensional shape of a folded polypeptide
is result of the interactions among the R-groups
 Protein Chemistry

Levels of Structural Organization

[3 Structure …]
Important Features:
1. Amino acid residues that are distant from each other
in the 1o structure come into close proximity when
the polypeptide folds.
2. When polypeptide folds, proteins become more
compact; most water molecules are excluded from
the proteins’ interior making interactions bet. polar
and nonpolar AA possible.
3. Large globular proteins (> 200 AA) often contain
several compact units called domains.
Domains are structurally independent segments that
have specific functions (e.g. binding an ion)
 Protein Chemistry

Levels of Structural Organization

[3 Structure …]

Types of Interactions that Stabilize Tertiary Structure

 Hydrophobic interactions
 Electrostatic interactions (salt bridges)
 Hydrogen bonds
 Covalent bonds (disulfide bridges)
 Protein Chemistry

Interactions that Stabilize Tertiary Structure

 Protein Chemistry

R-R Interactions that maintain tertiary structure

 Protein Chemistry

Myoglobin
 Protein Chemistry

Levels of Structural Organization

 Protein folding is assisted by molecular
chaperones and enzymes

 Molecular chaperones – proteins that have the

net effect of increasing the rate of correct folding
by binding newly synthesized polypeptides
before they are completely folded.
[e.g., heat-shock proteins, chaperonins]
 Enzymes involved in protein folding
1. Peptidyl prolyl cis-trans isomerase
2. Protein-disulfide isomerase
 Protein Chemistry

Levels of Structural Organization

D. Quaternary Structure
- exhibited only by proteins containing more than
one polypeptide chain.
- most proteins with molecular masses > 100 kD,
consist of more than one polypeptide chain.
* Each polypeptide component is called a subunit.

- Oligomeric proteins are those with more than

one subunit.
* Subunits maybe identical or different.
* Protomers are identical subunits.
 Protein Chemistry

Levels of Structural Organization

[Quaternary …]

 Quaternary structure describes the

characteristic manner in which the
individual , folded polypeptide chains fit
each other or interact with one another so
that they can act as one single molecule.
 Protein Chemistry

Hemoglobin Structural organizations of keratin

 Protein Chemistry

Levels of Structural Organization

[Quaternary …]
Interactions that hold subunits together
 Hydrophobic interactions
 Electrostatic interactions
 Hydrogen bonds
 Interpolypeptide disulfide bonds

 Hydrophobic interactions are the principal

forces that hold the subunits together.
 Electrostatic forces contribute to the proper
alignment of the subunits.
 Protein Chemistry

Protein Denaturation
Denaturation refers to the loss of function of the
protein when it losses its 3-dimensional structure.

 under most conditions, denatured proteins exist in

a set of partially folded states.
 denaturing forces disrupt the weak interactions in
a protein, primarily hydrogen bonds.
 cleavage of noncovalent bonds results to
disruption of the 2, 3 and quaternary structures
 Protein Chemistry

Protein Denaturation
 Chemical, physical & biological alterations result
from denaturation
 Denaturing agents include:
Physical [heat, extremes of pH, UV, surface action,
high pressure…]
Chemical [organic solvents,(H) (alcohol, acetone),
solutes like(D) urea, or by(H) detergents]
 Certain globular proteins regain their native
structure and biologic activity if returned to
conditions in which the native conformation is
stable – a process known as renaturation.
 Protein Chemistry

Protein Purification

 precedes protein analysis

 Fractionation procedures – to eliminate

selectively the other components of the
mixture so that only the required protein
remains.
 Protein Chemistry

Protein Purification
Characteristics of proteins that are used in the various
separation procedures

Characteristics Procedure
Charge Ion exchange chromatography
Electrophoresis
Polarity Hydrophobic interaction
chromatography
Size Gel filtration chromatography
SDS-PAGE
Ultracentrifugation
Binding specificity Affinity chromatography
 Protein Chemistry

Protein Purification
Chromatography

 Process/technique for fractionation,

isolation, purification and identification of
substances in a mixture based on the
relative affinities of component mixtures
between 2 phases: mobile phase and
stationary phase
Gel Filtration Chromatography Affinity Chromatography
 Protein Chemistry

Protein Analysis
Steps in Protein analysis:

I. Determine amino acid composition of the

protein
II. Determine the N and C terminals
III. Determine the amino acid sequence
IV. Check number of polypeptide chains
V. Identification of component amino acid
 Protein Chemistry

Steps in Protein Analysis…

I. Determine amino acid composition of the protein
[Acid hydrolysis; Basic hydrolysis; Enzymatic hydrolysis]
 Hydrolysis of proteins by acids:

- Disadvantages:
1. All the Trp & variable amounts of Ser & Thr are
destroyed
2. Gln and Asn are deamidated to Glu and Asp
3. Glu undergoes intramolecular dehydration to form
pyrollidone-5-carboxylic acid
4. Other amino acids may undergo intermolecular
dehydration forming cyclic anhydrides
 Protein Chemistry

Steps in Protein Analysis…

I. Determine amino acid composition of the protein
 Basic Hydrolysis :
Disadvantages:
1. Cysteine, cystine, Ser, Thr and Arg are decomposed
in the process
2. Other amino acids may be partially destroyed by
deamination

 Principal use of basic hydrolysis: To estimate

 Protein Chemistry

Steps in Protein Analysis…

I. Determine amino acid composition of the protein
 Enzymatic hydrolysis :

1. Pepsin – cleaves off peptide bonds whose amino

function is contributed by Phe, Tyr, Trp, Leu, Glu,
Gln
2. Trypsin – cleaves off peptide bonds whose carbonyl
function is contributed by Lys and Arg
3. Chymotyrpsin – cleaves off peptide bonds whose
carbonyl function is contributed by Phe, Trp and Tyr
4. Carboxypeptidase – cleaves off C-terminal amino
acids
 Protein Chemistry

Steps in Protein Analysis…

I. Determine amino acid composition of the protein
 Enzymatic hydrolysis :

5. Aminopeptidase – cleaves off N-terminal AA

6. Thermolysin – heat-stable bacterial protease;
cleaves off peptide bonds whose carbonyl function is
contributed by Leu, Val and Ile
7. Papain – found in certain plants like papaya; cleaves
off peptide bonds whose carbonyl function is
contributed by Lys, Leu, Arg, Gly
8. Bromelain – derived from pineapple; cleaves off
peptide bonds whose carbonyl function is contributed
by Lys, Ala, Tyr and Gly
 Protein Chemistry

Steps in Protein Analysis…

II. Determine N and C terminals
 N-terminal:
1) Sanger’s reaction
2) Dansyl chloride reaction
3) Edman’s reaction
4) Reaction with aminopeptidase

 C-terminal:
1) Reduction with lithium borohydride
2) Hydrazinolysis
3) Reaction with carboxypeptidase
 Protein Chemistry

Steps in Protein Analysis…

III. Determine amino acid sequence

 Edman’s reaction:
- liberates amino acids one at a time from the
N-terminus of a polypeptide. The N-terminal
residue is removed as a phenyl-thiohydantoin
derivative.
- carried out on a programmed machine called
sequenator.
 Protein Chemistry

Steps in Protein Analysis…

IV. Check number of polypeptide chains

 If there are 2 or more chains, separate them.

A. Disulfide bonds
- oxidation with performic acid
- reduction with mercaptans

B. Noncovalent bonds
- denaturing agents
 Protein Chemistry

Steps in Protein Analysis…

V. Identification of component amino acids

1. Paper chromatography
Solubility,hydrophobic organic solvent, H2O
molecule bound to the cellulose

2. Thin layer chromatography

Glass plates for support
3. Ion exchange chromatography -
Cation exchanger - polysulfonic; low to high pH
Anion exchanger – polyamino; high to low pH

4. Electrophoresis
will move at cathode(-),anode(+) or stationary

5. High performance liquid chromatography

 KWASHIORKOR AND MARASMUS –
PROTEIN (polypeptide) DEFICIENCY.
 SCURVY – IMPAIRED FORMATION OF THE
AMINO ACIDS HYDROXYPROLINE AND
HYDROXYLYSINE.
 MENKE’S SYNDROME – DEFECT (cofactor
lost) IN POLYPEPTIDE CROSS-LINKING
(Lysyl Oxidase).
 OSTEOGENESIS IMPERFECTA – DEFECT IN
PROTEIN (collagen) BIOSYNTHESIS.
 EHLER’S – DANLOS SYNDROME –
ENZYMATIC DEFECT (Lysyl Hydroxylase) IN
AMINO ACID BIOSYNTHESIS.