PROTEINS

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PHARMACEUTICAL BIOCHEMISTRY LECTURE

PHA 6112

UNIT II: PROTEINS

OUTLINE

2.1 General Structural Characteristics of Proteins

2.2 Structures of Proteins
q Primary Structure
q Secondary Structure
q Tertiary Structure
q Quaternary Structure
2.3 Protein Hydrolysis
2.4 Protein Denaturation
2.5 Classification of Proteins
2.6 Disease Associated with Proteins

INTENDED LEARNING OUTCOMES

q Discuss the different interactions that give rise to the four levels of protein structure
q Predict the effects of pH, temperature and chemical agents on the organization of protein
structure
q Give examples of proteins and relate their structure to their function
q Explain the molecular basis of some disease states
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2.1 GENERAL STRUCTURAL CHARACTERISTICS OF PROTEINS

q General definition - Naturally-occurring, unbranched polymer in which the monomer units

are amino acids

q Specific definition - Peptide in which at least 40 amino acid residues are present
• The terms polypeptide and protein are used interchangeably to describe a protein
• Several proteins have >10,000 amino acid residues
• Common proteins contain 400–500 amino acid residues
• Small proteins contain 40–100 amino acid residues

• Based on Polypeptide Chain Present

• Monomeric: Protein which contains one polypeptide chain
• Multimeric: Protein which contains two or more polypeptide chains

o One kind of chain: homomultimer

o >1 kind of chain: heteromultimer
o hemoglobin is a heterotetramer composed of 2 α-chains and 2 β-chains
§ Hetero – α and β chains
§ Tetra – 2 α-chains + 2 β-chains

• Based on Chemical Composition

• Simple protein: Protein in which only amino acid

residues are present

• Conjugated protein: Protein that has one or more non-

amino-acid entities (prosthetic groups) present in its
structure
o One or more polypeptide chains may be present
o Non-amino-acid components may be organic or
inorganic

• May be classified further based on the nature of prosthetic group(s) present

o Lipoprotein contains lipid prosthetic groups
o Glycoprotein contains carbohydrate groups
o Metalloprotein contains a specific metal as its prosthetic group

• Based on Shape
• Fibrous proteins – α-keratin & collagen
• The polypeptide chains are arranged in long strands or
sheets
• Long rod-shaped or string-like molecules that can
intertwine with one another and form strong fibers that
are water insoluble.
• Structural functions
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• Globular proteins – myoglobin & hemoglobin

• The polypeptide chains are folded into spherical
or globular shapes
• Nonpolar AAs are in the interior, polar AAs are on
the exterior.
• Water soluble character allows movement
through the blood and other body fluids to sites
where their activity is needed.
• Dynamic functions

• Based on Function
• Catalytic proteins
• Defense proteins
• Transport proteins
• Messenger proteins
• Contractile proteins
• Structural proteins
• Transmembrane proteins
• Storage proteins
• Regulatory proteins
• Nutrient proteins

• Based on Amino Acid Contents

• COMPLETE PROTEINS contains the essential AA in the proper amounts

• INCOMPLETE PROTEINS is low in one or more of the essential amino acids,

usually lysine, tryptophan or methionine.
• Proteins from animal sources are complete, except gelatin
• Proteins from vegetable sources are incomplete, except soy protein

• COMPLEMENTARY PROTEINS are incomplete proteins which when served

together complement each other and provide all the essential amino acids

2.2 PROTEIN STRUCTURE

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q Primary Structure
• Primary structure: Order in which amino acids are linked together in a protein
• Every protein has its own unique amino acid sequence
• Frederick Sanger sequenced and determined the primary structure for the first
protein (insulin) in 1953

• Order in which amino acids are linked together in a protein through peptide bonds.
• It is distinctive of a protein (or polypeptide) and tells its AA composition.
• It defines the structure, shape and function of the protein.
• Starts with N-terminal (left) side to C-terminal (right)
• The sequence is dictated by the DNA base sequence gene. Errors in the DNA may
result to erroneous, non-functional protein.

• Primary structure of a specific protein is the same within the organism

• Structures of certain proteins are similar among different species of
animals
• Example: Insulin from pigs, cows, sheep, and humans are
similar but not identical

• Peptide linkages are essentially planar, 6 atoms lie in the same plane (C=O, C-N and
N-H)
• Planar peptide linkage structure is rigid, thus rotation of C-N group is hindered; cis-
trans isomerism is possible (the trans being highly favored)
• The effect is peptide bond planarity resulting to zigzag arrangement with a protein
backbone
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q Secondary Structure
• The ordered 3D arrangements or regular folding in localized regions of a polypeptide chain
• Spatial arrangement of the atoms in the polypeptide chain
• Formed and stabilized by H-bond between the amide proton and carbonyl O.
• The primary structure dictates the secondary structure.
• The only bond responsible for the secondary structure of proteins is H-bonding between
peptide bonds, the –C=O of the one peptide group and the –N-H of another peptide linkage
farther along the backbone.

• Alpha Helix
• Single protein chain resembling coiled spring (helix) by H bonds
• H-bonding between AA within the same chain (intramolecular H-bonding)
• R group stay outside the helix because there is not enough space for them to stay inside.
• The helix is tightly wound that the space in the center is too small for solvent molecules to enter.
• Must have the same conformations (all D or all L) or will not coil

• Beta-pleated Sheet
• “Pleated” or zigzag pattern
• Completely extended protein chain segments in same or different molecules governed by
intermolecular (between molecules) or intramolecular (within the molecule) H-bonding
• R or side chains are below or above the sheet and backbone is alternating top and bottom position.
• U-turn structure is the most frequently encountered.
• Intermolecular H-bonding can be
• Parallel –chains run in the same direction
• Antiparallel –chains run in opposite direction which makes it more stable because of
fully collinear H-bonds.
• Unstructured segments
• Portions of protein with neither alpha helix or beta pleated sheet structure
• Confers flexibility to proteins, i.e. they interact with several different substances.
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q Tertiary Structure
• The overall 3D shape of a protein
• Results in interactions between AA side chains that are widely separated from each other.
• This defines the biological function of the proteins.

• Proteins may have, either the two forms:

• Fibrous (insoluble) – mechanical strength, structural components, movement
• Globular (soluble) – transport, regulatory, enzymes
• In general 4 types of interactions are observed
• Disulfide bonding
• Electrostatic interactions
• H-bonding
• Hydrophobic interactions

INTERACTIONS NATURE OF BONDING

Hydrophobic Interactions
Hydrophilic Interactions
Electrostatic
Interactions/Salt Bridges
Hydrogen Bonds
Covalent Disulfide Bonds
Interactions between non-polar groups
Attractions between polar or ionized groups and water
on the surface of tertiary structure
Ionic interactions between ionized acidic and basic
amino acids
Occur between H and O or N
Strong covalent links between sulfur atoms of two
cysteine amino acids

q
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q Quaternary Structure
• Refers to organization among the various polypeptide chains in a multimeric protein
• Highest level protein organization
• Present only in proteins that have 2 or more polypeptide chains (subunits).
• Subunits are generally independent of each other (not covalently bonded).
• Aka oligomeric proteins

SUMMARY of PROTEIN STRUCTURES

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2.3 PROTEIN HYDROLYSIS

• Reverse of peptide bond formation
• Results in the regeneration of an amine and carboxylic acid functional groups
• Protein digestion - Enzyme-catalyzed hydrolysis
• Free amino acids produced are absorbed into the bloodstream and transported to the
liver for the synthesis of new proteins
• Hydrolysis of cellular proteins to amino acids is an ongoing process, as the body resynthesizes
needed molecules and tissue
• Complete hydrolysis
• All peptide bonds are broken freeing all AAs; products are free AAs
• Partial hydrolysis
• Some peptide bonds are broken; products are free AAs and small peptides

• Protein digestion is simply enzyme-catalyzed hydrolysis of ingested proteins

• The free amino acids produced from this process are absorbed through the intestinal wall into
the bloodstream and transported to the liver
• In the liver, they become raw materials for the synthesis of new protein tissue
• Also, the hydrolysis of cellular protein to amino acids is an on-going process, as the body re-
synthesizes needed molecules and tissue.

2.4 PROTEIN DENATURATION

•
Partial or complete disorganization of a protein’s characteristic 3D shape (disordered)
•
Loses biochemical activity
•
Does not affect primary structure of proteins
•
Sometimes denaturation can be reversed = renaturation (“refolded”), but usually is
irreversible
• Loses water solubility – usual property of denatured proteins
• Precipitation of denatured proteins = coagulation
Applications
• “Cooked” proteins are easily digested because it is easier for digestive enzymes to act on
denatured proteins
• Cooking also kills microorganisms also through protein denaturation
• Ham & bacon harbors parasites that can cause trichinosis
• Cauterization = In surgery, heat is used to seal small blood vessels
• Heating of surgical instruments for sterilization
• Patient’s body temperature should not exceed 106°F(41°C) as body enzymes begin to
inactive à lethal effects
• UV from sun causes denaturation (sunburn)
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• RENATURATION
o Restoration process in which protein is “refolded”
o Only very few proteins undergo renaturation.
o Denaturation is irreversible in most proteins.
• Two Types of Denaturing Agents:
o Physical: heat, high pressure, UV rays, agitation
o Chemical: acids, bases, organic solvents salts of heavy metals
• Consequences of Denaturation
o Physical: decreased solubility; precipitation
o Chemical: increased viscosity
o Biological: loss of hormonal and/or enzymatic activity

2.5 CLASSIFICATION OF PROTEINS

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2.5.3 According to Biological Roles

1. Structural protein
• Serve as supporting filaments, cables with sheets to give biological structures strength and
protection
a. Collagen: fibrous protein found in tendons and cartilage with high tensile strength
o leather: almost pure collagen
b. Elastin: structural protein capable of stretching in two dimensions
o ligaments
c. Keratin: largely tough insoluble protein found in hair, fingernails and feathers
d. Fiborin: found in silk fibers and spider webs
e. Resilin: has perfect elastic property found in wing hinges of some insects
f. Glycoproteins: acts as cell membrane and cell coats
g. Mucoprotein: mucous membrane, synovial fluids
h. Sclerotin: exoskeleton of insects
2. Catalytic Protein/Enzymes
• Most varied and most highly specialized proteins with catalytic activity
• Serves to increase rate of thousands of chemical reactions
• Biochemical reactions involve exchange of electrons between atoms of reacting molecules
which is being hastened by the presence of enzymes
o Enzymes are protein catalysts, capable of enhancing the rates of reactions by factors
of up to 1014. For example, the enzyme carbonic anhydrase catalyzes the reaction:
CO2 + H2O ↔H+ + HCO3-
by a factor of 107 over the uncatalyzed reaction.
o Alcohol Dehydrogenase: important in alcohol fermentation
o Arginase: hydrolyzes arginine
o Ribonuclease: hydrolyzes RNA
o Urease: hydrolyzes urea to form CO2 + H2O
o Cytochrome C: responsible for the transport electrons
o Chymotrypsin: cleaves the polypeptide chain from the C terminal of Arginine and Lysine
o Trypsin: cleaves the polypeptide chain from the N terminal of Arginine and Lysine
o Rennin: digestive enzyme which acts on casein
o Aminopolypeptidase: sequentially cleaves the protein chain from the N terminal

3. Nutrient and Storage proteins

• Seeds of many plants store nutrients (proteins required for growth of the germinating
seedling)
• Eg., seed proteins of wheat, corn and rice
a. Ovalbumin: major protein in egg white
b. Casein: major protein in milk
c. Ferritin: stores iron in the spleen
d. Gliadin: seed storage in wheat
e. Zein: seed storage in corn
f. Thymus histone: stored in thymus gland
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4. Transport proteins
• These bind and carry specific molecules and ions from one organ to another
• Many small molecules are transported both inside and outside cells when bound to
carrier proteins. Examples include the oxygen transport and storage proteins hemoglobin
and myoglobin, respectively. The proteins in membranes often permit the passage of
both small and large molecules through membranes.
• Binds to and carries specific molecules or ions from place to place
• Hemoglobin: carry oxygen from lungs to body organs
• Β1-lipoproteins: carry lipids from the liver to the other organs
• Hemocyanin: transport oxygen in the blood of some invertebrates
• Myoglobin: transport oxygen in the muscles
• Serum albumin: transports fatty acids
• Transportin: transports steroids
• Transcobalamin: transports vitamin B12
• Transferrin/Siderophilin/Iron-Binding Globulin: transports iron
• Cytochrome C: transports electrons
• Ceruloplasmin: transports copper in the blood

5. Contractile proteins
• Provides cells and organelles with the ability to contract, to change shape and to move
about
o Actin: thin and moving filaments of the myofibril in skeletal muscles;
o Myosin: thick and stationary filament in skeletal muscles; moving filaments of the
myofibril
o Tribulin: protein from which microtubules are built; they act in concert with other
proteins in flagella and cilia to propel cells.
o Kinesin moves protein cargoes around cells along microtubule “rails” formed by
tubulin, which is also present in the flagella of sperm cells.

6. Toxins
• Clostridium botulinum: causes bacterial food poisoning
• Diphtheria toxin: bacterial toxin
• Snake venoms: contains enzymeswhich hydrolyze phosphoglycerides
• Ricin: toxic protein of castor bean

7. Regulatory Proteins: help regulate cellular and physiological activity

• Hormones: product of living cells that circulates in body fluids or sap and produces a
specific effect (stimulatory) on the acitivity of cells remote from its point of origin
o Insulin: regulates blood glucose level
o Growth hormones: control growth of bones
o Adrenocorticotrophic hormone: regulated corticosteroid synthesis

8. Antibody/Protective proteins
• Defend organisms against invasion by other species and protect them from injury
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o Immunoglobulins/Antibodies
§ Specialized proteins made by the lymphocytes of vertebrates
§ Released by plasma cells to fight infection
§ Recognize and inactivate invading bacteria
§ Can recognize and precipitate or neutralize invading bacteria, viruses, or
foreign protein from other species
ü Macroglobulin (IgM): natural antibody, first to fight infections but is short-
lived for it cannot pass through the placenta
ü Classic Antibody (IgG): takes over the IgM in continued infection
ü Immunosurface protection antibody (IgA): present in all body secretions
most especially breast milk
ü Regain/Homocytotrophic Antibody (IgE): increased during allergic
reactions like asthma, pneumonia, hay fever
ü IgD: structure and function still unknown
• Complement: forms complexes with some antigen-antibody systems
• Fibrinogen: precursor of fibrin in blood clotting
• Thrombin: component of the clotting mechanism

2.6 DISEASES ASSOCIATED WITH PROTEINS

2.6.1 Collagen
Properties and Function
• Found in skin, bones, tendon, cartilage and teeth
• Most abundant protein in mammals
• Water-insoluble fibers
• Has great tensile strength
• Function: stress-bearing of connective tissues
Structure
• Contains repeating X-Y-Gly (where X is any amino acid; Y is often Pro or hyroxyproline); Lys, 5-
hydroxylysine and His residues are present at some of the X & Y in the triplet repeat
• Has unusual amino acids:
• 4-hydroxyproline
• 3-hydroxyproline
• 5-hydroxylysine
• Pro and HyPro make up 30% of residues
• Each polypeptide chain has about 1,000 AA residues
Disease
• In the synthesis of collagen, Vitamin C is required as cofactor for enzymes prolyl hydroxylase and
lysyl hydroxylase to function.
• These enzymes are responsible for the hydroxylation of Pro and Lys in collagen.
• Impaired hydroxylation of Pro and Lys results to collagen instability and the connective tissue
problem seen in scurvy (deficiency in Vit C).
•
2.6.2 Elastin
• A protein that gives connective tissue its elastic properties-like rubber band.
• Found in lungs, walls of large blood vessels and elastic ligaments
• Skin to allow skin to stretch and then spring back to shape.
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• Vessels and heart to stretch to control blood pressure.

• In joints to allow cartilage to absorb shock and avoid injury.
• Consists predominantly of nonpolar AA residues 1/3 Gly, 1/3 Val + Ala, rich in Pro and produces a
random coil conformation
• Elastin fibers associate by desmosine crosslinks (formed by 3 modified Lys and 1 Lys residues)
• Can be stretched to several times their normal length but recoil to original shape when relaxed.
• Deletion of the elastin gene was found in approx. 90% of patients with Williams Syndrome

2,.6.3 Myoglobin
• Made up of a single polypeptide chain with 153 AA; 8 alpha-helices; globular protein
• Has a single heme group
• Has high affinity for oxygen
• Primary function: oxygen storage protein, abundant in skeletal muscles
• Tetrameric protein made up of 2 α and 2 β subunits both with 153 AAs.
• Each monomer is similar to myoglobin structure.
• Each monomer has 1 heme. Thus can bind up to 4 oxygen molecules.
• Primary function: Oxygen transport protein

SICKLE CELL ANEMIA

• Sickle or crescent shape RBC
• Substitution of Val to Glu on the 6th aa of β subunit
• Change in the 10 structure produce hydrophobic patch on the surface of Hb
• hydrophobic patch interacts with other hydrophobic patches causing the Hb to aggregate into
strands that align into insoluble fibers
• Less efficient in delivering O2

2.6.4. Insulin
• Produced from β-cells of Islets of Langerhans in the pancreas
• Made up of 2 polypeptide chains (51 AAs); with inter- and intra-chain disulfide linkage
• It promotes glucose intake from blood into fat, liver and skeletal muscle cells.
• Hypoglycemic hormone
• Glucose in the cell is converted to glycogen (glycogenesis) or fats (lipogenesis).

• DIABETES MELLITUS
• Type 1: failure of the pancreas to produce enough insulin
• Type 2: a condition in which cells fail to respond to insulin (insulin resistance)

2.6.5 Glucagon
• Produced from α-cells of Islets of Langerhans in the pancreas
• Made up of single polypeptide chain (29 AAs)
• It elevates the blood glucose level by promoting synthesis of glucose (gluconeogenesis) and
breakdown of glycogen into glucose molecules (glycogenolysis).
• Hyperglycemic hormone
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2.7 Antibody
• Also known as immunoglobulins (Ig)
• Secreted mostly by the differentiated B lympocytes (plasma cells).
• Ig is a glycoprotein.
• Y-shaped molecule consists of 2 identical Light chains (~25kDa) and 2 identical Heavy chains
(~50kDa) held together by disulfide bonds.
• Each chain contains constant and variable regions. - The variable regions in Ab serve as binding
sites to the epitope of the antigen.

• IgA – found in mucosal areas, such as the gut, respiratory tract and urogenital tract. Prevents
colonization by pathogens. Also found in saliva, tears and breast milk.
• IgD – found mainly as antigen receptor on B cells that have not been exposed to antigens. It has
been shown to activate basophils and mast cells to produce antimicrobial factors.
• IgE – binds to allergens and triggers histamine release from mast cells and basophils, involved in
allergy. Also protects against parasitic worms.
• IgG – has 4 forms. Provides the majority of antibody-based immunity against invading pathogens.
This is the only Ig that can cross the placenta to give passive immunity to the fetus.
• IgM – expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very
high avidity. Eliminates pathogens in the early stages of B cell-mediated (humoral) immunity before
there is sufficient IgG.

• Can distinguish “self” from “non-self” molecules.

• Ab can tag a microbe or an infected cell for attack by other parts of the immune system, or
• Ab can neutralize its target directly (for example, by blocking a part of a microbe that is essential for
its invasion and survival).
• Auto-immune disease – presence of self-reactive immune response. Body “fights” against self.

REFERENCES:
• Stoker, H. Stephen, (2017). Biochemistry 3rd ed., Cengage learning STOKER
• Campbell, Harper Voet, D., Voet, J.G., Pratt C.W. (2013). Principles of biochemistry 4th ed.
Singapore pte. Ltd.: John
• Murray et.al, Harper’s Biochemistry 26th edition
• Lehninger’s Principles of Biochemistry 4th Edition