PROTEIN STRUCTURE

AND FUNCTION
 Proteins
 Greek work proteios meaning “first”
 Proteins play a crucial role in biological processes
 Proteins are polymers of amino acids
 Classification of Proteins based on
A. FUNCTION

1. Enzymes are biological catalysts. The majority of the enzymes

that have been studied are proteins. Reactions that would take
days or weeks or require extremely high temperatures without
enzymes are completed in an instant. For example, the
digestive enzymes pepsin, trypsin, and chymotrypsin break
downproteins in our diet so that subunits can be absorbed for
use by our cells.
 Important biological functions

2. Defense proteins include

antibodies (also called
immunoglobulins ) which are
specific protein molecules
produced by specialized cells of
the immune system in response
to foreign antigens. These
foreign invaders include bacteria
and viruses that infect the body.
Each antibody has regions that
precisely fit and bind to a single
antigen. It helps to end the
infection by binding to the antigen
and helping to destroy it or
remove it from the body.
 Important biological functions

3. Transport proteins carry materials from one place to another in the

body.
 The protein transferrin transports iron from the liver to the bone marrow,
where it is used to synthesize the heme group for hemoglobin. The proteins
hemoglobin and myoglobin are responsible for transport and storage of
oxygen in higher organisms, respectively.
 Important biological functions

4. Regulatory proteins control many aspects of cell function, including

metabolism and reproduction. We can function only within a limited set of
conditions. For life to exist, body temperature, the pH of the blood, and
blood glucose levels must be carefully regulated. Many of the hormones
that regulate body function, such as insulin and glucagon, are proteins.
adrenaline
http://www.danzettwoch.com/howthatworks/images/zettwoch_adrenaline.jpg
 Important biological functions

5. Structural proteins provide mechanical support to large animals and

provide them with their outer coverings.
 Our hair and fingernails are largely composed of the protein keratin. Other
proteins provide mechanical strength for our bones, tendons, and skin.
Without such support, large, multicellular organisms like ourselves could not
exist.
epidermolysis bullosa and the “butterfly babies”

People with Dystrophic EB are missing key structural proteins that

connect their two upper layers of skin.
 Important biological functions

6. Movement proteins are necessary for all forms of movement. Our

muscles, including that most important muscle, the heart, contract and
expand through the interaction of actin and myosin proteins. Sperm can
swim because they have long flagella made up of proteins.
 Important biological functions

7. Nutrient proteins serve as sources of amino acids for

embryos or infants. Egg albumin and casein in milk are
examples of nutrient storage proteins.
 The a-Amino Acids
 As the name implies, these compounds contains both an
amine and an acid
 Hundreds are formed both naturally and synthetically
 Only 20 are common in nature
 All 20 are a-amino acids
 Alpha means the amine is adjacent to the carboxylate
group
 19 out of the 20 are stereoisomers
 Amino Acids

 a-carbon is attached to a:
 Carboxylate group
 Protonated amino group

 At physiologic pH the amino acid has:

 Carboxyl group in –COO-
 Amino group in –NH3+

 Neutral molecule with equal number of + and –

charges is a zwitterion
Zwitterions
 Amino acids are white crystalline solids with high
melting points and high water solubilities
 The two charged groups, the basic amino group and
the carboxylic acid, at the two ends lead to internal
proton transfer – zwitterions
 By changing the pH you can affect the net charge on
the zwitterions
 The pH point at which there is no net charge on the
zwitterions is called the isoelectric point (pI)
Stereoisomers of Amino Acids

 The a-carbon of
amino acids is chiral
 Glycine is the only
common amino acid
that is not chiral
 Amino acid
configuration isolated
from proteins is L-
 Draw most oxidized
end of the molecule,
carbonyl, at the top
 Classes of Amino Acids
 All differences between amino acids depend upon their
side-chain R groups
 Form amino acid classes based on the polarity of their
side chains
 Polar, neutral have a high affinity for water, but are not
ionic at pH 7
 Negatively charged have ionized carboxyl groups in their
side chains
 Positively charged are basic as the side chain reacts with
water to release a hydroxide anion
Names of Common Amino Acids
Essential amino acids:
 all amino acids are essential for normal tissue
growth and development
 the term is reserved for those amino acids that must
be supplied in the diet for proper growth and
development . The other amino acids can be
synthesized from carbohydrates and lipids in the
body by enzyme-catalyzed reactions if a source of
nitrogen is also available.
Essential amino acids:
 must be supplied in the diet because either that
there are no biochemical pathways available for
their synthesis or the available pathway do not
provide adequate amounts for proper nutrition.
 His, Met, Arg, Thr, Trp, Ileu, Leu, Lys, Val, Phe
(semi-essential; only on infancy stage)

 PVT. TIM HALL – Phe, Val, Thr, Trp, Ileu, Met, His,
Arg, Leu, Lys
Non-Polar (Hydrophobic) Amino Acids
Polar (Hydrophilic) Neutral Amino Acids
Charged Amino Acids

Acidic Basic
Amino Acids as Acids and Bases
Amino Acids as Acids and Bases
16.3 Draw the structure of alanine at pH=1, 6.02, and 12
Electrophoresis and charged amino acids

Electrophoresis is an analytical method for identifying amino

acids by observing their migration as a function of pH under an
applied electric field gradient.

- paper or gel (polyacrylamide)

- Saturated with buffer solution
- Solution of unknown amino AA is placed at the center of
paper
- Electrodes are connected to the ends of the paper and
electric current is allowed to pass through the sol’n
- Ninhydrin is sprayed (reacts with Aas to produce colored
products, green/blue)
Electrophoresis and charged amino acids

The amino acid carries a

POSITIVE CHARGE at pH<pI
and migrates to the NEGATIVE
ELECTRODE (cathode)

The amino acid carries a

POSITIVE CHARGE at pH>pI
and migrates to the POSITIVE
ELECTRODE (anode)
Follow-Up Problem

1. To which electrode will alanine migrate in electrophoresis at

pH=1, 6.02, and 12?
Follow-Up Problem

2. An unknown, containing some combination of ALANINE,

LYSINE, or ASPARTIC ACID, is subjected to paper
electrophoresis at pH=7. Ninhydrin treatment shows some
amino acid at the negative electrode and some amino acid
has not moved from the center. No amino acid is found at
the positive electrode. What AA(s) is (are) in the unknown?
The Peptide Bond
 Proteins are linear polymers of L-α-amino acids
 Carboxyl group of one amino acid is linked to the amino group
of another amino acid
 Linkage is an amide bond or peptide linkage

 This reaction is a dehydration reaction as water is released

Dipeptides
 Condensing or dehydrating two amino acids produces a
dipeptide
 Amino acid with a free a-NH3+ group is the amino terminal amino
acid, N-terminal for short
 Amino acid with a free -COO- group is the carboxyl terminal amino
acid, C-terminal for short
 Amino acid structures are written with the N-terminal on the left
 amino acids are polymerized into peptides and
proteins; in general, protein is used for molecules
composed of over 50 amino acids; peptide is used
for molecules of less than 50 amino acids
 Oligopeptide – 10-20 Aas
Naming peptides

The far right AA residue retains the name of the

amino acid

All AAs (except TRYPTOPHAN)  -ine or –ic acid

is replaced by –yl

Tryptophan becomes tryptophanyl

Writing the Structure of Peptides
Writing the Structure of Peptides
Writing the Structure of Peptides
Estimating the pI of Peptides
 The pI of a peptide containing only NEUTRAL Aas is in
the range of pI values of neutral Aas, 5.05 to 6.30

 For peptides with basic and acidic residues, the pI is

approx. the average of the pI values of the acidic and
basic Aas.

 Phe-Asp-Ala has only the acidic Asp, pI of peptide = pI

of Asp, 2.77
Some examples of biochemically important
small peptides:

1. Aspartame (Asp-Phe)
Sold under the trade names
Nutrasweet and Equal,
aspartame is the artificial
sweetener used in almost
every diet food on the market
today. Its caloric content is the
same as sucrose but is ~180
times as sweet. Both AAs
present in the dipeptide must
be in the L- form for the sweet
taste to occur; the L-D, D-L,
and D-D forms have a bitter
taste.
Some examples of biochemically important
small peptides:

1. Glutathione (Glu-Cys-Gly)
The tripeptide, produced by the body itself, is present in
significant concentrations in most cells and is of
considerable physiological importance as a regulator of
oxidation-reduction reactions. It functions as an
antioxidant, protecting cellular contents from oxidizing
agents such as peroxides and superoxides, which are
highly reactive forms of oxygen often generated within a
cell. Glu is bonded to Cys through the side-chain
carboxyl group rather than through the α-carbon
carboxyl group.
 Some examples of biochemically important
small peptides:

3. Enkephalins (Tyr-Gly-Gly-Phe-Leu & Tyr-Gly-Gly-Phe-Met)

Enkephalins and endorphins - natural painkillers produced in the

body; bind to receptors in the brain to give relief from pain.
This effect appears to be responsible for the runner’s high, for
the temporary loss of pain when severe injury occurs, and for the
analgesic effects of acupuncture.
Β-endorphins and runner’s high
Oxytocin stimulates uterine contractions in labor and
vasopressin is an antidiuretic hormone that regulates blood
pressure by adjusting the amount of water reabsorbed by
the kidneys.
The sickled cells are unable to pass through the small capillaries
of the circulatory system, and circulation is hindered. This results
in damage to many organs, especially bone and kidney, and can
lead to death at an early age.
 The Primary Structure of Proteins
 Primary structure is the amino acid sequence of the
polypeptide chain
A result of covalent bonding between the amino acids – the
peptide bonds
 Each protein has a different primary structure with
different amino acids in different places along the
chain
Resonance and Rigidity of Peptide Bonds
There is free rotation
around only two
of the three single
bonds of a peptide
backbone.
R groups on adjacent amino acids are on opposite sides of the
chain because of the rigid peptide bond.
 The Secondary Structure of Proteins
 When the primary sequence of the polypeptide folds
into regularly repeating structures, secondary
structure is formed
 Secondary structure results from hydrogen bonding
between the amide hydrogens and carbonyl oxygens
of the peptide bonds
 Not all regions have a clearly defined secondary
structure, some are random or nonregular
 a-Helix
 Most common type of secondary structure
 Coiled, helical
 Important features:
 Each amide H and carbonyl O is involved in H bonds locking the
helix in place
 Carbonyl O links to amide H 4 amino acids away

 H bonds are parallel to the long axis of the helix

 Helix is right-handed

 Repeat distance or pitch is 5.4 angstroms

 3.6 amino acids per turn

a-Helix
a-Helices in Fibrils
 Fibrous proteins are
arranged in a
secondary structure of
fibers or sheets with
only 1 type of
secondary structure
 Repeated coiling of
helices
 b-Pleated Sheet

 Second most common secondary structure appears similar to folds

of fabric
 All of the carbonyl O and amide H are involved in the H bonds
with the chain nearly completely extended
 b-Pleated Sheet

 Two possible orientations

 Parallel if the N-termini are head-to-head
 Antiparallel if the N-terminus of one chain is aligned with the C-terminus of
the other
18.6 Tertiary Structure
 The three-dimensional structure, which is distinct from
secondary structure is classified as tertiary structure
 Globular tertiary structure forms spontaneously and is
maintained by interactions among the side chains or R
groups
 Tertiary structure defines the biological function of
proteins
Types of Interactions Maintaining Tertiary Structure

 Disulfide bridges between two cysteine residues

- the only covalent bond, the strongest of the 3o bonds; link
chains together and cause chains to twist and bend
- a covalent bond between 2 S atoms formed by the oxidation
of the – SH groups on two cysteine amino acids
- S – S linkage can occur within the same chain (intrachain), or
between 2 or more chains (interchain), or both inter- and
intrachain
Types of Interactions Maintaining Tertiary Structure

 Salt bridges (ionic interaction/ electrostatic attraction)

between ionic side chains -COO- and -NH3+

- some amino acid side chains may contain positively charged

groups, others negatively charged groups. If properly positioned
these groups may give rise to bonding between different
portions of given molecule, or between 2 or more protein chains

 Hydrogen bonds between polar residue side chains

 Hydrophobic interactions: two nonpolar groups are attracted
by a mutual repulsion of water
Types of Interactions Maintaining Tertiary Structure
Interactions Involved in Tertiary Structure
18.7 The Quaternary Structure of Proteins
 The functional form of many proteins is not that of a single
polypeptide chain, but actually an aggregate of several
globular peptides
 Quaternary structure: the arrangement of subunits or peptides
that form a larger protein
 Subunit: a polypeptide chain having primary, secondary, and
tertiary structural features that is a part of a larger protein
 Quaternary structure is maintained by the same forces which
are active in maintaining tertiary structure
Conjugated Proteins
Class Prosthetic Group Example
Nucleoprotein Nucleic acids Viruses

Lipoprotein Lipids Serum lipoproteins

Glycoprotein Carbohydrates Mucin in saliva

Phosphoprotein Phosphate groups Casein in milk

Hemoprotein Heme Hemoglobin,

cytochormes
Metalloprotein Iron, zinc Ferritin, hemoglobin
18.8 An Overview of Protein Structure and Function

Types of Protein Structure and Their

Interrelationships
1. Primary structure:
 Amino acid sequence
 Results from formation of covalent peptide bonds
between amino acids
2. Secondary structure:
 Includes α-helix and β-sheet
 Hydrogen bonding between amide hydrogens and
carbonyl oxygens of the peptide bonds
Types of Protein Structure and Their
Interrelationships

3. Tertiary structure:
 Overall folding of the entire polypeptide chain
 Interactions between different amino acid side chains
4. Quaternary structure:
 Concerned with topological, spatial arrangement of two or
more polypeptide chains
 Involves both disulfide bridges and noncovalent interactions
 Protein Functions Follow Shape

 Fibrous proteins:
 Mechanical strength
 Structural components

 Movement

 Globular proteins:
 Transport

 Regulatory

 Enzymes
Important Peptides and Protein Hormones
Name Origin Action
Adrenocorticotropic hormone (ACTH) Pituitary Stimulates production of adrenal

Angiotensin II Blood Plasma Cause blood vessels to constrict

Follicle-stimulating hormone (FSH) Pituitary Stimulates sperm production and

folicle maturation
Gastrin Stomach Stimulates stomach to secrete acid
Glucagon Pancreas Stimulates glycogen metabolism
in liver
Human Growth Hormone ( HGH) Pituitary General effect: bone growth
Insulin Pancreas Controls metabolism of
carbohydrates
Oxytocin Pituitary Stimulates contraction of uterus
and other smooth muscles
Prolactin Pituitary Stimulates lactation
Somatostatin Hypothalamus Inhibits production of HGH
Vasopressin Pituitary Decreases volume of urine
excreted
 18.9 Myoglobin and Hemoglobin
Myoglobin and Oxygen Storage
 Hemoglobin is the oxygen-
transport protein of higher
animals
 Myoglobin is the oxygen

storage protein of skeletal

muscle
 Oxygen is transferred from

hemoglobin to myoglobin as
myoglobin has a stronger
attraction for oxygen than
hemoglobin does
Heme and Oxygen Binding
 Heme group is an
essential component of
the proteins hemoglobin
and myoglobin
 Fe2+ ion in the heme
group is the oxygen
binding site
 Each hemoglobin contains
a heme group which can
hold 1 molecule of
oxygen
 18.20 Denaturation of Proteins
 Extremes of temperature and pH have a drastic
effect on protein conformation
 Denaturation
 Is the loss of organized structure of a globular
protein
 Does not alter primary structure
 Protein Hydrolysis
76

Protein hydrolysis
 splits the peptide bonds to give smaller

peptides and amino acids

 occurs in the digestion of proteins

 occurs in cells when amino acids are needed to

synthesize new proteins and repair tissues
 Hydrolysis of a Dipeptide
77

 In the lab, the hydrolysis

of a peptide requires
acid or base, water,
and heat.
 In the body, enzymes
catalyze the hydrolysis
of proteins.
 Denaturation
78

Denaturation involves
 the disruption of bonds in the secondary, tertiary, and
quaternary protein structures
 heat and organic compounds that break apart H bonds

and disrupt hydrophobic interactions

 acids and bases that break H bonds between polar R

groups and disrupt ionic bonds

 heavy metal ions that react with S—S bonds to form
solids
 agitation, such as whipping, that stretches peptide

chains until bonds break

Some practical aspects of protein denaturation

1. Heat and UV
- cooking denatures proteins ; e.g., egg white proteins have
to be denatured by cooking for them to become utilizable by
our system
- any burn, including sunburn, causes denaturation of
protein in the body
- sterilization uses UV and heat in the form of steam to
coagulate the proteins of bacteria
2. Salts of heavy metal ions esp. Hg2+, Pb2+, Ag+
- used as antiseptics in low concentrations, in higher concentrations
they act as poisons. When ingested they precipitate the proteins in
cells of body tissues. Effective treatment consists of feeding with
egg white, followed by an emetic (the egg white forms complex
with the poison and taken out of circulation by emetic)

3. Organic compounds such as soap, detergents, phenol, and

aliphatic alcohol
- the hydrophobic portions of these compounds interact with the
hydrophobic core of the protein, while the hydrophilic portion is H-
bonded with the aqueous environment. This causes swelling and
concomitant unfolding of the protein molecules.
18.21 Sequencing a Protein

The procedure in the determination of amino acid

sequence basically involves the following steps:

1) hydrolysis - can be effected by acid, alkali, or

enzyme

2) identification of the products of hydrolysis

3) fitting the pieces together as you would a jigsaw

puzzle
18.21 Sequencing a Protein
HYDROLYSIS

A) Acid Hydrolysis
- involves heating in the presence of 6N HCl in sealed tube at
110oC for 10 – 100 hrs. depending on the nature of peptide
or protein to be hydrolyzed
- the protein is completely hydrolyzed, but trp, is destroyed
completely and ser, thr, and tyr are partially destroyed
18.21 Sequencing a Protein
HYDROLYSIS

B) Alkaline Hydrolysis
- heating in the presence of 4N NaOH in sealed tube at 10 –
100 hrs as in acid hydrolysis
- does not damage trp, but destroys arg, cys, thr, & ser;
and some amino acids are partly deaminated
- more disadvantageous but since it does not destroy trp, it
is used in quantitative determination of this amino acid
C) Enzymatic Hydrolysis
- by proteases/ peptidases

1)Exopeptidases – enzymes that cleave

external peptide bonds

a) Aminopeptidases
- sequentially cleaves peptide bonds, beginning at the N-
terminal end of the polypeptide
- the liberated amino acids are identified one by one
b) Carboxypeptidases
- sequentially cleaves peptide bonds beginning at the C-
terminal end of the polypeptide

The exopeptidases may be used in the determination of the

amino acid sequence of peptides. By following the increase in
amino acid liberated during hydrolysis by the two enzymes it is
possible to determine the amino acid sequence in a peptide.
EXAMPLE:

A time-course analysis of the free amino acid in the

hydrolysate of the pentapeptide with aminopeptidase and
carboxypeptidase gave the following results:

a) with carboxypeptidase
[Glu] > [Val] > [Ala] > [Cys] = [Arg] ; cyst and arg appear
simultaneously on equal amounts
b) with aminopeptidase
[Arg] > [Cys] > [Ala] > [Val] = [Glu] ; val and glu appear
simultaneously on equal amounts
c) what is the primary structure of the pentapeptide?
Other methods of determining the N-terminal end
(chemical method)

1. 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 reacts with the amino
nitrogen of the N-terminal amino acid.

HF
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.

H3 O+
 Other methods of determining the N-terminal end
(chemical method)

2. Edman Degradation

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.

 The key reagent in the

Edman degradation is
phenyl
isothiocyanate.
 Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.

(labeling)

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.

phenylthiocarbamoyl (PTC)
(releasing) HCl derivative

thiazolone Peptide shortened

by 1 residue
Under the conditions of its formation, the thiazolone rearranges to
a phenylthiohydantoin (PTH) derivative.

Thiazolone

PTH-derivative
The PTH derivative is isolated and
identified. The remainder of the
peptide is subjected to a second
Edman degradation.
Other method of determining the C-terminal end
(chemical method)

1. Hydrazine Method

hydrazine reacts with all amino acids whose carboxyl

group is bound in peptide linkage, creating amino acyl
hydrazides. Only the C-terminal amino acid is spared.

2. Cyanogen Bromide (CNBr) – cleaves peptide bonds

at the C-end of methionine
Other method of determining the C-terminal end
(chemical method)

2. Hydrazine Method

hydrazine reacts with all amino acids whose carboxyl

group is bound in peptide linkage, creating amino acyl
hydrazides. Only the C-terminal amino acid is spared.
ENDOPEPTIDASES
cleaves internal peptide bonds
1. Trypsin

cleaves peptide bonds at the carboxyl end of the two

strongly basic amino acids: arginine and lysine
2. Chymotrypsin

cleaves peptide bonds at the carboxyl end of the three

aromatic amino acids: phenylalanine, tyrosine, &
trptophan; and Leucine
3. Elastase

cleaves on the carboxyl side of Gly and Ala

 4. Pepsin

cleaves peptide bonds at the amino end of the

three aromatic amino acids: phenylalanine,
tyrosine, tryptophan; acidic amino acids, Asp and
Glu; and Ileu
 5. Thermolysin

cleaves peptide bonds at the amino end of the three

aromatic amino acids, Phe, Tyr, Trp; and amino acids
with bulky nonpolar R groups, Leu, Ileu, and Val
Example:

Write the peptides generated from chymotrypsin,

trypsin, and pepsin digestion of

Ala-His-Tyr-Pro-Trp-Arg-Ileu-Phe-Glu-Lys-Cys